Heterogeneity of the DN4 (CD44-CD25-) subset of CD4-CD8- double negative thymocytes; dependence on CD3 signaling.

Recent studies have shown that apoptotic cell death associated with selection for thymocytes that express clonotypic TCRbeta or TCRgammadelta proteins takes place in the DN4 (CD44-CD25-) subset of CD4-CD8- double negative (DN) thymocytes. A detailed analysis of the DN4 subset is therefore of interest. Using intracellular (IC) staining for clonotypic TCR and CD3varepsilon proteins we find that DN4 cells consist of five subpopulations: TCRbetaIC(high)/CD3varepsilonIC(high)/TCRgammadeltaIC-, TCRbetaI-C-/CD3varepsilonIC(high)/TCRgammadeltaIC(+), TCRbetaIC(high)/CD3varepsilonIC(high)/TCRgammadeltaIC(+), TCRbetaIC(low)/CD3varepsilonIC(low)/TCRgammadeltaIC(-), and TCRbetaIC(-)/CD3varepsilonIC(-)/TCRgammadeltaIC(-). Expression levels of IC TCRbeta/CD3varepsilon, and of Thy1.2, CD2, and CD69 at the cell surface suggest that the TCRbetaIC(low)/CD3varepsilonIC(low)/TCRgammadeltaIC(-) subset harbors the direct precursors of DP cells, and is critical for life/death decisions in early thymic selection. TCRbeta/CD3varepsilon downregulation is less pronounced in DN4 and DP cells of mice deficient for CD3zeta or for p56(lck), suggesting that the dynamics of TCR protein regulation in the DN4 subset is dependent on CD3 signaling.