In Situ Hybridization of Interferon‐Gamma Producing Peripheral Blood Mononuclear Cells

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Abstract
Individual interferon‐gamma (IFN‐gamma) producing cells in activated human peripheral blood mononuclear cells (PBMC) were characterized by in situ hybridization using [35S]‐labelled antisense RNA probes. The proportion of positive cells expressing IFN‐gamma mRNA varied according to the substances used for stimulation. IFN‐gamma mRNA expressed a relatively low percentage of 1 8% PBMC after a single stimulus with mitogens or OKT‐3 antibody and 20‐30% of the cells were identified to synthesize IFN‐gamma mRNA after stimulation with PHA + P‐MA+OKT‐3 antibody. The expression of IFN‐gamma mRNA and production of the lymphokine was dependent on accessory cells. If accessory ceils were replaced by recombinant interleukin‐1 (IL‐l) plus interleukin‐6 (IL‐6). then T‐cell proliferation to phytohaemagglutinin (PHA) could be partially restored and measurable amounts of IFN‐gamma were detected. The addition of interIeukin‐2 (IL‐2) or phorbol‐12‐myrislate‐13‐acetate to T cells stimulated with PHA. IL‐1 and IL‐6 did not restore the production of IFN‐gamma to an extent comparable to that produced by T cells stimulated in the presence of accessory cells. In further studies, depletion of T‐cell subsets showed that CD3+, CD4+, CD8+, CD29+ and CD45RA+ cells were involved in IFN‐gamma production after mitogenic stimulation. In conclusion, our data demonstrate that IFN‐gamma production is dependent on signals from accessory cells and IFN‐gamma is synthesized by only a small proportion of T cells, that did not belong to a unique population, characterized by conventional cellular surface antigens.