Single cell analysis of cytokine gene coexpression during CD4⁺ T-cell phenotype development

CD4⁺ T cells from αβ-T-cell receptor transgenic mice were analyzed for coexpression of cytokine mRNAs during phenotype development using a double- label in situ hybridization technique. T cells that produced cytokines in the primary response were a fraction of the activated population, and only a minority of the cytokine-positive cells coexpressed two cytokines. In secondary responses, frequencies of double-positive cells increased, although they remained a minority of the total. Of the cytokine pairs examined, interleukin (IL)-4 and IL-5 were the most frequently coexpressed. IL-4 and interferon γ showed the greatest tendency toward segregation of expression, being rarely coexpressed after the primary stimulation. These data indicate that there is significant heterogeneity of cytokine gene expression by individual CD4⁺ T cells during early antigenic responses. Coexpression of any pairs of cytokines, much less Th1 and Th2 cytokines, is generally the exception. The Th0 phenotype is a population phenotype rather than an individual cell phenotype.