Figure 2. (A) Analysis of CD3e and CD3eM protein tyrosine phosphoryla- tion after stimulation of thymocytes with pervanadate. Thymocytes (108) from nontransgenic B6 mice (N.Thy), CD3eD/D; e-tg (e-tg), and CD3eD/D; eM-tg (eM-tg) mice were incubated for 5 min in medium lacking (2) or con- taining (1) pervanadate. Cell lysates were immunoprecipitated with anti- CD3e (mAb 145-2C11). Immunopre- cipitated proteins were resolved by 12% SDS-PAGE, followed by antiphospho- tyrosine (mAb 4G10) Western blotting (top blot). Blots were subsequently stripped and reprobed with anti-CD3e (mAb HMT3.1; bottom blot). (B) Ge- netic reconstitution of mice deficient for z and CD3e (z2/2 3 eD/D) with trans- genes encoding WT or mutant CD3e proteins. Two-color plots show CD4/ CD8 staining profiles on total thy- mocytes or total lymph node cells. Numbers in quadrants indicate the per- centage of cells within that quadrant. Histograms show CD3 surface staining on total thymocytes or total lymph node cells. Total thymocyte numbers are pro- vided in the histograms. Results are shown for two independently generated eM-tg founder lines with similar trans- gene copy number. face expression and/or pre-TCR signaling is compromised in the absence of TCR z chain. When mice rendered defi- cient for both CD3e and TCR-z (eD/D 3 z2/2) were re- constituted with the WT CD3e transgene, a phenotype es- sentially indistinguishable from that of z2/2 mice was observed (i.e., low numbers of DP and SP thymocytes and peripheral T cells), reflecting the ability of the transgenic CD3e protein to rescue the eD/D but not the z2/2 defect (Fig. 2 B). However, two independently generated eM transgenes expressing similar (z13) levels of eM protein were unable to effect a similar partial rescue of T cell devel- opment when introduced into eD/D 3 z2/2 mice (Fig. 2 B). In addition to demonstrating that the eM protein is signal- ing defective in vivo, these results reveal that a signaling threshold exists for the pre-TCR, below which this com- plex is incapable of inducing the formation of DP thy- mocytes. These findings are consistent with those of Ar- douin et al. (24) demonstrating a gene dosage effect of CD3e for pre-TCR function in the absence of z chain. As both pre-TCR surface expression and signaling are thought to be compromised in z2/2 mice, a more complete under- standing of pre-TCR signaling requirements awaits the analysis of eD/D 3 z2/2 mice reconstituted with signaling- deficient forms of both proteins. 916 CD3e-mediated Signals in T Cell Development