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  • Title: Distinct pattern of IL-2 and IFN-gamma gene expression in CD4 and CD8 T cells: cytofluorometric analysis at a single cell level using non-radioactive probes.
    Author: Morvan PY, Picot C, Dejour R, Genetet B, Genetet N.
    Journal: Cell Mol Biol (Noisy-le-grand); 1995 Nov; 41(7):945-57. PubMed ID: 8595373.
    Abstract:
    IL-2 and IFN-gamma gene expression was analyzed using an original method for in situ hybridization (ISH) with non-isotopic probes and flow cytometric analysis (FC). This method permits rapid detection of mRNA at a single cell level among in vitro activated human peripheral blood mononuclear cells (PBMC) and purified CD4 and CD8 T cell subsets. After stimulation with PMA and ionomycin (PMA+Io), cells were fixed at different times and hybridized with digoxigenin (DIG)-labelled RNA antisense or sense probes specific for IL-2 and IFN-gamma. The level of cytokine gene expression in individual cells was visualized using FITC-conjugated anti-DIG antibodies and the fluorescent signal was analyzed by flow cytometry. Specific hybridization with IL-2 and IFN-gamma antisense probes was detected among activated PBMC within lymphoid cells identified by their light scattering properties. Kinetic analysis of the frequency of mRNA producing cells exhibited a biphasic pattern with an early peak at 6-8 hrs. when percentages of IL-2 and IFN-gamma expressing cells reached 35 +/- 7% and 18 +/- 4%, respectively. Similar data were obtained by enzymatic detection on cell smears using AP-conjugated anti-DIG. Combination of ISH with FC was applied to the comparison of the pattern of cytokine gene expression between CD4 and CD8 T cell subsets isolated by negative selection using immunobeads and magnetic separation. IL-2 was expressed by activated CD8 T cells (25-35%), but CD4 T cells were the major producers of IL-2 as assessed by the high frequency of mRNA expressing cells (60%) and the large amount of mRNA per cell relative to the mean fluorescence intensity. In contrast, IFN-gamma mRNA was preferentially expressed by CD8 T cells (27-37%) and a minority of CD4 T cells (17-23%). Despite quantitative differences, kinetic analysis of IL-2 gene expression in CD4 and CD8 T cells showed similar profiles with an early peak at 6-8 hrs. Upregulation of IL-2 gene expression in CD4 T cells by CD28 co-stimulation increases the amount of IL-2 mRNA per cell as visualized by mean fluorescence intensity. In addition the effect of CD28 co-stimulation on IL-2 mRNA stabilisation was demonstrated by the maintenance of a high frequency of IL-2 expressing CD4 T cells and an elevated level of mRNA per cell for prolonged period after PMA+Io stimulation. By contrast CD28 co-stimulation had no obvious effect on IFN-gamma expression.
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