Recent studies have shown that apoptotic cell death associated with selection for thymocytes that express clonotypic TCRβ or TCRγδ 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 CD3ε proteins we find that DN4 cells consist of five subpopulations: TCRβIChigh/CD3εIChigh/TCRγδIC-, TCRβI-C-/CD3εIChigh/TCRγδIC+, TCRβIChigh/CD3εIChigh/TCRγδIC+, TCRβIClow/CD3εIClow/TCRγδIC-, and TCRβIC-/CD3εIC-/TCRγδIC-. Expression levels of IC TCRβ/CD3ε, and of Thy1.2, CD2, and CD69 at the cell surface suggest that the TCRβIClow/CD3εIClow/TCRγδIC-subset harbors the direct precursors of DP cells, and is critical for life/death decisions in early thymic selection. TCRβ/CD3ε downregulation is less pronounced in DN4 and DP cells of mice deficient for CD3ζ or for p56lck, suggesting that the dynamics of TCR protein regulation in the DN4 subset is dependent on CD3 signaling. © 2002 Elsevier Science B.V. All rights reserved.
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