Immature thymocytes that fail to express TCR beta and/or TCR gamma delta proteins die by apoptotic cell death in the CD44(-)CD25(-) (DN4) subset

Pre-TCR/CD3 signals are essential for survival and maturation of (CD44(-)25 (+)) DN3 thymocytes via the (CD44(-)25(-)) DN4 stage to CD4(+)CD8(+) (DP) c ells, a process termed beta -selection. The exact developmental stages of a poptosis resulting from lack of pre-TCR/CD3 signals have so far not been de termined. Here we analyzed apoptotic cell death in relation to expression o f clonotypic TCR polypeptides and to cell cycle status in immature thymocyt e subpopulations of wild type (wt) mice and of several strains of mice with compromised pre-TCR/CD3 signaling complexes. In wt mice or pre-TCR/CD3-def icient mice, apoptotic cells could not be detected among DN3 cells but accu mulated in a subset of DN4 expressing CD69. Apoptotic CD69(+)DN4 cells were rare in wt mice and were found among DN4 cells that were negative or low f or intracellular TCR beta and negative for TCR gamma delta polypeptide chai ns. Apoptotic CD69(+)DN4 cells were abundant in pre-TCR/CD3 signaling-defic ient mice in which most DN4 cells failed to express clonotypic TCR polypept ides. Survival of DN4 cells, but not maturation of DN3 cells to DN4, was fo und to depend on the expression of clonotypic TCR polypeptides in the same cell. The results suggest that thymocytes unsuccessful in alpha beta or in gamma delta lineage development die by apoptosis in the DN4 subset.