Intracellular cytokine production by human CD4(+) and CD8(+) T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) induced in human CD4(+) and CD8(+) T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20 h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4(+) cells can make IL-2 than the other two cytokines, and that there are more TNF-alpha-positive CD4(+) cells than cells with IFN-gamma. For normal CD8(+) cells the highest production of cytokine is of IFN-gamma (up to 50% of the cells) especially at longer rimes (10-20 h) of stimulation. For CD8(+) cells, IL-2-positive cells exceed those with TNF-alpha. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20 h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4(+) or CD8(+) cells. Finally using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-alpha but significantly raised levels of production of IFN-gamma in both CD4(+) and CD8(+) lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.