DECREASED FREQUENCY OF INTERFERON-GAMMA-PRODUCING AND INTERLEUKIN-2-PRODUCING CELLS IN PATIENTS WITH ATOPIC DISEASES MEASURED AT THE SINGLE-CELL LEVEL

Background: Recently, diminished interferon-gamma (IFN-gamma) and incr eased interleukin (IL)-4 production in peripheral blood mononuclear ce lls (PBMCs)) from atopic patients have been described by several group s, measured as total cytokine content in culture supernatants. These s tudies suggested a predominance of T-H2-like cells producing large amo unts of IL-4 in atopic patients. It is not clear whether the reported cytokine imbalances are the result of an alteration in the distributio n of specific T-cell subsets or whether intrinsic dysregulation in cyt okine production is a characteristic of atopic individuals. Objective: This study examined the production of IFN-gamma, IL-4, and IL-2 in PB MCs from atopic patients at the single cell level with the use of fres hly isolated lymphocytes. Methods: We recently described a flow cytome tric assay in which three-color analysis was used to study the product ion of a cytokine of interest in a T-cell subpopulation defined by two cell surface markers. PBMCs fram 23 atopic patients and 14 control su bjects were stimulated with phorbol ester and ionomycin for 5 hours. P BMCs from seven patients and seven control subjects were also cultured with immobilized anti-CD3 antibodies for 24 hours Cells were fixed ma de permeable, and stained for intracellular cytokines in combination w ith cell surface markers CD3, CD8, and CD45RO. Cytokine;producing cell s were analyzed by gating on T-cell subsets. Results: IFN-gamma-produc ing cells were significantly decreased (p < 0.05) in CD4+ T cells but not in CD8+ T cells of atopic patients. CD45RO+ and CD45RO- T cells sh owed a decreased proportion of IFN-gamma-producing cells (p < 0.05 and p < 0.01, respectively). IL-2-production was diminished in all T-cell subsets (p < 0.01). The number of IL-4-producing cells was not elevat ed and such cells were exclusively found in the CD45RO+ T cells. Analy sis of culture supernatants of sorted CD45RO+ T cells for IL-4 and IFN -gamma production confirmed these results. Conclusion: Out findings pr ovide evidence that a reduced IFN-gamma production in atopic patients is due to an intrinsic defect selectively found in the CD4+ T cells Be cause IL-2 production was markedly decreased but IL-4 production was u nchanged, our data demonstrate a deficiency in the ability of atopic T cells to produce T-H1-like cytokines on stimulation with phorbol este r; ionomycin, or anti-CD3 monoclonal antibodies.