human T lymphocytes in vitro examined using a double immunocytochemical technique

A number of studies suggest that cytokines may contribute to the pathogenesis ofviral infections, including dengue. In this study, we developed a dou- ble immunocytochemical method and characterized cytokine-producing cells in the peripheral blood mono- nuclear cells (PBMC) of dengue virus-immune donors after inritro sthnulation with specffic dengue antigens. We found that double immunostaining using immuno- alkaline phosphatase (Vector blue) for cytokines linter- feron-'y (IFN-y), interleukin (IL) -2, -4, -la, -1�3, and -6, tumor necrosis factor �3 (TNF-(3), and TNF-aJ and immunoperoxidase Itliaminobenzi����e (DAB)1 for cell surface markers (CD3, CD4, CD8, CD2O, and CD68) provided the best distinction of double-positive cells from single-positive or -negative cells. The number ofWN-'y, IL-2, IL-4, and TNF43-positive cells increased 2 or 3 days after stimulation with specffic dengue anti- gens. No or very few cytokine-producing cells were detected in the PBMC of non-immune donors stim- ulated with dengue antigens and the PBMC of im- mune donors stimulated with a control antigen. The analysis of cell surface markers showed that mainly CD4� and CD8� T cells produced these cytokines. The results obtained by immunocytocheniistry were consistent with cytokine levels detected in the culture medium assayed by enzyme-linked immunosorbent assay. hi conclusion, this double iznmunocytochemistry technique is suitable for the detection and character- ization ofcytokine-producing cells in PBMC. Further- more, the results support the hypothesis that antigen- sthnulated CD4� and CD8� T cells produce cytokines that may play a role in the pathogenesis of dengue virus infection. J. Leukoc. Biol. 61: 338-345; 1997.