Detection by flow cytometry of T cell subsets secreting IL-2 and UFNy(Gamma): optimalisation for the technic and the establishment of reference values

• Published in: Pathologie biologie (Paris) Vol. 55; no. 5; p. 222
• Main Authors: Kallel, C, Masy, E, Duthilleul, P
• Format: Journal Article
• Language: French
• Published: France 01.06.2007
• Subjects: Adult; Antigens, CD - analysis; CD4-Positive T-Lymphocytes - immunology; Cell Culture Techniques; Cell Membrane - immunology; Cytokines - metabolism; Flow Cytometry - methods; Fluorescein-5-isothiocyanate; Humans; Interferon-gamma - secretion; Interleukin-2 - secretion; Ionomycin - pharmacology; Kinetics; Reference Values; T-Lymphocyte Subsets - cytology; T-Lymphocyte Subsets - drug effects; T-Lymphocyte Subsets - immunology; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0369-8114
• DOI: 10.1016/j.patbio.2006.02.007

A dysregulation in Th1/Th2 balance has been described for different pathological situations. Knowing the cytokine profile in a given pathology could assist in understanding the disease mechanism and in choosing an immune intervention most effective for the management of this condition. In this work, the production of two Th1 cytokines, IL-2 and IFN- gamma, was analyzed for different T-cell subsets from 20 normal subjects (mean age 33.5 years) and reference values were defined using the flow cytometric analyses. The optimum operating conditions were set as following: mononuclear cells were stimulated with PMA (20 ng/ml) and ionomycin (1 uM) for 6 h in the presence of brefeldin A (10 ug/ml). Cells were fixed with 4% paraformaldehyde and then dually stained, with anti-CD3 or anti-CD4 or anti-CD8 for the membrane and with anticytokine antibody for the intracytoplasma after being permeabilized with 0.5% saponine solution. The frequency determination of cells that produce IL-2 or IFN-gamma revealed large 95% confidence intervals: (CD3-IL-2: 4.60-10.67%, CD8-IL-2: 1.47-23%, CD3-IFN-gamma: 2,97-32,49%, CD4-IFN-gamma: 2.83-21%, CD8-IFN-gamma: 4.60-35.28%). CD4+ lymphocytes produce the majority of IL-2 (85 vs 13% for CD8+). For IFN-gamma, the situation is more balanced, but the CD4+ lymphocytes remain the predominant producer cells (63 vs. 41%).