Regulation of Zap70 Expression During Thymocyte Development Enables Temporal Separation of CD4 and CD8 Repertoire Selection at Different Signaling Thresholds

Regulated expression of Zap70 determines whether thymocytes develop into CD4+ or CD8+ T cells. Timing Is Everything As they develop into mature CD4+ or CD8+ single-positive (SP) T cells in the thymus, immature thymocytes undergo positive selection, during which they have both CD4 and CD8 [double-positive (DP) cells] and their T cell antigen receptor (TCR) is stimulated by self-peptide–major histocompatibility complexes. It remains unclear whether it is the strength or the duration of TCR signaling that determines how a cell “chooses” to become a CD4+ or CD8+ SP cell (see the Perspective by Alarcón and van Santen). Saini et al. generated transgenic mice that expressed an inducible gene encoding the tyrosine kinase Zap70, a critical mediator of TCR signaling, in a Zap70-deficient background (TetZap70 mice). Thymocytes in Zap70-deficient mice are arrested at the DP stage because of defective TCR signaling, but this block was removed by induction of Zap70 expression in the TetZap70 mice, after which CD4+ SP cells developed more quickly than did CD8+ SP cells. The temporal difference in SP cell development, also seen in wild-type mice, occurred because CD4+ SP cells required less Zap70 protein to develop than did CD8+ SP cells. In addition, this study showed that DP thymocytes were heterogeneous, with CD4+ and CD8+ SP cells arising from different DP subpopulations. Together, these data suggest that regulation of the abundance of Zap70 enables the temporal differential development of CD4+ and CD8+ cells, which is based on signal strength through the TCR, thus incorporating both the “strength” and the “duration” models of positive selection. To investigate the temporal regulation of the commitment of immature thymocytes to either the CD4+ or the CD8+ lineage in the thymus, we developed a transgenic mouse that expressed a tetracycline-inducible gene encoding the tyrosine kinase ζ chain–associated protein kinase of 70 kD (Zap70), which restored development in Zap70−/− thymocytes arrested at the preselection, CD4+CD8+ double-positive (DP) stage. After induction of the expression of Zap70 and the production of Zap70 protein, CD4+ single-positive (SP) cells that expressed Zbtb7b (which encodes the CD4+ T cell–associated transcription factor ThPOK) became abundant within 30 hours, whereas CD8+ SP cells were not detectable until day 4. We found that mature CD4+ and CD8+ cells arose from phenotypically distinct subsets of DP thymocytes that developed with different kinetics and contrasting sensitivities to stimulation of the T cell antigen receptor (TCR). In wild-type mice, expression of endogenous Zap70 progressively increased during maturation of the DP subsets, and the abundance of Zap70 protein determined the sensitivity of the cells to stimulation of the TCR. This temporal gradient in the amount of Zap70 protein enabled the selection of CD4+ and CD8+ repertoires in separate temporal windows and at different TCR signaling thresholds, thereby facilitating discrimination of distinct positive selection signals in these lineages.