IN-SITU HYBRIDIZATION AND CYTOFLUOROMETRIC ANALYSIS OF CYTOKINE MESSENGER-RNA DURING IN-VITRO ACTIVATION OF HUMAN T-CELLS

We present an original method for in situ hybridization (ISH) using no n isotopic probes and flow cytometry analysis that permits rapid detec tion of lymphokine transcripts at single cell level in an in vitro act ivated human Jurkat T cell line and in peripheral blood T Cell subsets . After PMA and either ionomycin or ConA stimulation, cells were fixed and hybridized with digoxigenin (DIG)-labelled RNA antisense or sense probes specific for IL-2 and IFN-gamma. The level of cytokine gene ex pression in individual cells was visualised using FITC-conjugated anti -DIG antibody, and the resultant signal was analysed by flow cytometry . IL-2 mRNA was first detected in activated Jurkat T cells. Addition o f cycloheximide 4 hours after the beginning of stimulation increased b oth the frequency of labelled cells and the amount of mRNA per cell, a s determined by the mean fluorescence intensity. The specificity and s ensitivity of IL-2 mRNA detection were tested by comparison with North ern blot analysis and in situ hybridization (ISH) with immuno-cytochem ical staining. IL-2 and IFN-gamma mRNA were detectable in PBMC as earl y as 3 hours after in vitro stimulation with PMA and ionomycin. The fr equency of positive cells and the amount of mRNA per cell peaked at 6- 8 hours, when the percentages of IL-2 and IFN-gamma mRNA-containing ce lls reached 30 - 40% and 15 - 20%, respectively. The two lymphokines w ere expressed in both CD4(+) and CD8(+) T cells, but the frequency of IL-2 expressing cells and the amount of IL-2 mRNA per cell were higher in CD4(+) (60 %) than in CD8(+) T cells (25 %), whereas IFN-gamma wer e preferentially transcribed by CD8(+) T cells (40 %). The results obt ained by this method were in accordance with the data obtained by Nort hern blot analysis, with cellular protein content estimated by immuno- fluorescence staining, and with IL-2 titration by bioassay. We compare d the performance of this method with ISH using radioactive probes.