Cytokine profile of clonal T cells from two hypereosinophilic patients. Total T cells (CD4⁺ and CD8⁺) were isolated from PBMC obtained from two hypereosinophilic patients and a healthy blood donor by negative selection. After a 6-hour culture (10⁶ cells/mL) with Brefeldine A (10 μg/mL) in the absence or in the presence of PMA (50 ng/mL) and A23187 (0.1 μg/mL), cell membranes were stained with FITC- or PE-conjugated anti-CD8 and PerCP-conjugated anti-CD3 MoAbs. The T cells were fixed and permeabilized before staining cytokines with PE- or FITC-conjugated MoAbs against IL-2, IFN-γ, IL-4, or IL-5. Intracytoplasmic expression of cytokines was analyzed by flow cytometry after gating on CD3⁻CD8⁻ (equivalent to CD3⁻CD4⁺) lymphocytes for patients and CD3⁺CD8⁻ (equivalent to CD3⁺CD4⁺) lymphocytes for the control subject. Filled histograms represent staining for cytokines in cells that have been stimulated with PMA and A23187. Negative controls (solid lines) are provided by unstimulated cells that have been incubated in the presence of Brefeldine A alone.