DISTINCT PATTERN OF IL-2 AND IFN-GAMMA GENE-EXPRESSION IN CD4 AND CD8T-CELLS - CYTOFLUOROMETRIC ANALYSIS AT A SINGLE-CELL LEVEL USING NONRADIOACTIVE PROBES

IL-2 and IFN-gamma gene expression was analyzed using an original meth od for in situ hybridization (ISH) with non-isotopic probes and flow c ytometric analysis (FC). This method permits rapid detection of mRNA a t 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 diff erent times and hybridized with digoxigenin (DIG)-labelled RNA antisen se or sense probes specific for IL-2 and IFN-gamma. The level of cytok ine gene expression in individual cells was visualized using FITC-conj ugated anti-DIG antibodies and the fluorescent signal was analyzed by flow cytometry. Specific hybridization with IL-2 and IFN-gamma antisen se probes was detected among activated PBMC within lymphoid cells iden tified by their light scattering properties. Kinetic analysis of the f requency of mRNA producing cells exhibited a biphasic pattern with an early peak at 6-8 hrs. when percentages of IL-2 and IFN-gamma expressi ng cells reached 35 +/- 7% and 18 +/- 4%, respectively. Similar data w ere 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 su bsets isolated by negative selection using immunobeads and magnetic se paration. IL-2 was expressed by activated CD8 T cells (25-35%), but CD 4 T cells were the major producers of IL-2 as assessed by the high fre quency of mRNA expressing cells (60%) and the large amount of mRNA per cell relative to the mean fluorescence intensity. In contrast, IFN-ga mma mRNA was preferentially expressed by CD8 T cells (27-37%) and a mi nority of CD4 T cells (17-23%). Despite quantitative differences, kine tic analysis of IL-2 gene expression in CD4 and CD8 T cells showed sim ilar 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. I n addition the effect of CD28 co-stimulation on IL-2 mRNA stabilisatio n was demonstrated by the maintenance of a high frequency of IL-2 expr essing CD4 T cells and an elevated level of mRNA per cell for prolonge d period after PMA+Io stimulation. By contrast CD28 co-stimulation had no obvious effect on IFN-gamma expression.