Figure 1. Phenotype of CD3eD/D; e-tg and CD3eD/D; eM-tg mice. Comparison of thymocytes (A) and lymph node cells (B) from control (e1/1), eD/D, eD/D; e-tg, and eD/D; eM-tg mice by FACS® analysis. Two-color plots show CD4/CD8 staining profiles. Numbers in quadrants indicate the percentage of cells within that quadrant. Histograms show CD3 surface staining on total thymocytes or total lymph node cells. Total thymocyte numbers are provided in the histograms. Results are representative of three separate experiments. (C) Analysis of CD42CD82 thymocyte subsets in CD3eD/D; e-tg and CD3eD/D; eM-tg mice. Left panels, two-color (CD25 vs. CD44) staining on gated (CD42CD82CD32) thymocytes. Right panels, CD3 vs. TCR-d staining on gated (CD42CD82) thymocytes. pre-TCR surface expression or for pre-TCR signaling re- sult in developmental arrest at this identical stage indicates that signaling by the pre-TCR is necessary for the matura- tion of DN, CD442CD251 thymocytes to the CD442 CD252 stage (1). Notably, DN, CD442CD252 thymo- cytes were present in eD/D; eM-tg mice in numbers similar to those observed in CD3eD/D; e-tg mice (Fig. 1 C), indi- cating that functional pre-TCR complexes are expressed in CD3eD/D; eM-tg mice. Finally, the mutant CD3e chain was also capable of rescuing the development of DN-a/b TCR1 and DN g/d TCR1 T cells when introduced into CD3eD/D mice, as these cells were detected both in the thy- mus (Fig. 1C) and periphery (data not shown) of CD3eD/D; eM-tg mice. The eM Transgene Encodes a Signaling-defective Protein. Based on the results of in vitro studies, the eM transgene is predicted to encode a protein that does not contribute to the TCR signaling response (4, 5, 20, 21). However, in view of the normal phenotype observed in CD3eD/D; eM-tg mice, we wished to examine this issue in greater detail in our transgenic system. A hallmark of ITAM function is its ability to become tyrosine phosphorylated upon T cell acti- vation, thereby creating a site for stable association with and activation of ZAP-70 (2). After activation with pervan- adate, tyrosine phosphorylation of both CD3e and TCR-z is observed in thymocytes from control (CD3e1/1) and CD3eD/D; e-tg mice (Fig. 2 A). In contrast, whereas per- vanadate stimulation of thymocytes from CD3eD/D; eM-tg mice induced tyrosine phosphorylation of TCR z chain, tyrosine phosphorylation of the mutated CD3e chain was nearly undetectable (Fig. 2 A). Similar results were ob- tained after stimulation of thymocytes by TCR cross-link- ing (data not shown). Reblotting with anti-CD3e mAb demonstrated the presence of the mutant CD3e protein in the immunoprecipitated CD3 complexes (Fig. 2 A). As the proximal ITAM tyrosine is retained in the eM protein, the extremely low level of tyrosine phosphorylation observed after stimulation was unexpected. Interestingly, a TCR z chain transgene containing Yﬁ F mutations of the NH2- (but not the COOH-) terminal tyrosine residues in each of its three ITAMs also failed to become phosphorylated in vivo upon T cell activation (9). Recent data suggest that tyrosine phosphorylation of the TCR z chain follows a hi- erarchical progression in which the phosphorylation of spe- cific ITAM tyrosines proceeds and perhaps facilitates phos- phorylation of other ITAM tyrosines (22). Our findings may reflect a similar hierarchy for phosphorylation of the CD3e-ITAM tyrosines. In any event, these results indicate that the eM protein is weakly (if at all) tyrosine phosphory- lated upon TCR engagement. Although a defect in thymocyte maturation was not ob- served in CD3eD/D; eM-tg mice, we reasoned that one might be revealed in the presence of another mutation that impairs pre-TCR/TCR surface expression and/or signal- ing. Mice lacking TCR z chain (z2/2) exhibit a profound, though incomplete, block in T cell development character- ized by reduced TCR surface expression and a reduction in the number of DP and SP thymocytes and mature, periph- eral SP T cells (reference 23; Fig. 2 B). The low number of DP thymocytes in z2/2 mice indicates that pre-TCR sur- 915 Sommers et al. Brief Definitive Report