response. CD3e is represented twice in the TCR complex, serving as a component of both the g/e and d/e dimers. In addition, after TCR cross-linking, CD3e and z are the pre- dominant tyrosine-phosphorylated TCR subunits. In this study, we assessed the importance of CD3e signals during T cell development by genetically reconstituting CD3e- deficient mice with transgenes encoding either the wild-type (WT) or a mutant (signaling defective) form of the protein. The results demonstrate that signals transduced by CD3e are not specifically required for T cell maturation but in- stead contribute quantitatively to TCR signaling. Interest- ingly, the phenotype of TCR-transgenic/CD3e-mutant mice suggests a potential role for CD3e signals in particular or TCR signals in general for the generation and/or sur- vival of mature T cells. Materials and Methods Generation of CD3eD/D, CD3eD/D; e-tg, and CD3eD/D; eM-tg Mice. The generation of mice lacking expression of CD3e (CD3eD/D) and huCD2-CD3e transgenic (tg) mice has been de- scribed previously (15). The huCD2-CD3eM transgene was gen- erated by mutating the murine CD3e ITAM sequence in vitro using a synthetic oligonucleotide that substitutes phenylalanine (AAA) for tyrosine (ATA) at position 181. Transgenic founder lines that expressed levels of protein that most closely matched that of endogenous CD3e were used in the experiments de- scribed here. TCR-transgenic mice used in these studies included the MHC class I–restricted TCRs H-Y (16) and P14 (17), which were maintained in the H-2Db background. Western Blot Analysis. Thymocytes were enumerated, washed twice in PBS, and resuspended in PBS at a concentration of 108 per milliliter. Thymocyte stimulations, immunoprecipitations, Western blotting, and polyacrylamide electrophoresis were per- formed as described (18, 19). Separated proteins were transferred to polyvinylidene difluoride membranes and blotted with antiphos- photyrosine mAb (4G10; Upstate Biotechnology) or monoclonal anti-CD3e (HMT3.1) followed by anti–mouse IgG–horserad- ish peroxidase (Transduction Laboratories) or protein A–horse- radish peroxidase (Transduction Laboratories), respectively, and detected by chemiluminescence (ECL; Amersham Pharmacia Biotech). Multicolor Flow Cytometry and Measurement of Calcium Flux. Single-cell suspensions of thymocytes or lymph node cells were processed, stained, and analyzed as described previously (8). Cal- cium flux measurements were performed as described (19). Proliferation and Cytokine Assays. Single-cell suspensions were prepared from thymi or lymph nodes in RPMI plus 10% FCS. CD41CD82 T cells were enriched by panning after incubating cells with antibodies to CD8, B220, and MAC-1 for 30 min. Al- ternatively, CD81CD42 thymocytes were enriched by panning after incubating cells with antibodies to CD4, B220, and MAC-1 for 30 min. Cells were washed, resuspended in RPMI plus 10% FCS, and placed on rabbit anti–mouse Ig–coated plates at 258C, 5% CO2. Nonadherent cells were collected after 1–2 h. In some experiments, purified cells were labeled with the intracellular flu- orescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) before stimulation. Cells were washed twice in HBSS, re- suspended at 4 3 106 cells/ml in HBSS and 50 ng/ml CFSE, in- cubated for 15 min at 378C, and then washed twice in HBSS and resuspended in RPMI plus 10% FCS. Accessory cells and APCs were prepared from spleen suspensions of C57BL/6 mice. APCs were depleted of T cells with anti-Thy1.2 1 C9 and irradiated with 3,000 rads. For antibody-mediated stimulation, wells of 96- well flat-bottomed plates were coated with antibodies at the indi- cated concentrations (see Fig. 3) overnight at 378C and washed three times with sterile media. 105 responder T cells were added to the wells, incubated at 378C for 40 h, and then pulsed with 1 mCi of [3H]thymidine for 8–12 h. For stimulation with peptide, 105 responder T cells were combined with 5 3 105 APCs (ob- tained from female mice) in flat-bottomed 96-well plates in the presence or absence of the peptide. Smcy peptide (KCSRN- RQYL) was synthesized in the FDA Core Facility and was added to culture at the indicated concentrations (see Fig. 3). Prolifera- tion and cytokine assays were performed as described (8, 19). For IL-2 production, cells were stimulated as described above and culture supernatants were harvested after 48 h. IL-2 ELISAs were performed using the reagents and protocol obtained from Phar- Mingen. Results and Discussion T Cell Development Is Restored in CD3eD/D; eM-tg Mice. T cell development is arrested at the CD42CD82 (DN) stage in CD3eD/D mice, such that the more mature subsets of CD41CD81 (DP), CD41CD82 (CD4-SP), and CD42CD81 (CD8-SP) thymocytes normally present in the thymus of adult mice are absent in CD3eD/D mice (Fig. 1 A; reference 15). As a consequence of this developmental block, total thymocyte numbers are markedly reduced in CD3eD/D mice (,10% of normal), and mature T cells are undetectable in the peripheral lymphoid organs (Fig. 1 A; reference 15). As reported previously (15) and shown in Fig. 1 A, expression of transgenic WT CD3e (e-tg) in CD3eD/D mice can rescue this developmental block, as as- sessed by the normalization of thymus cellularity and the presence of normal numbers of DP thymocytes and ma- ture, TCRhi SP T cells in the thymus and periphery. Sig- nificantly, genetic reconstitution with a transgene encoding a mutant form of CD3e that contains a Yﬁ F mutation of the distal tyrosine residue within the single CD3e ITAM (eM-tg) also rescued the developmental defect in CD3eD/D mice (Fig. 1 A). CD3eD/D; e-tg and CD3eD/D; eM-tg mice contained similar numbers of thymocytes, and these were composed of approximately the same ratio of DP, CD4- SP, and CD8-SP cells as control, CD3e1/1 mice (Fig. 1 A). In addition, normal numbers of TCRhi CD4-SP and TCRhi CD8-SP T cells were present in the periphery of CD3eD/D; eM-tg mice (Fig. 1 B). The normalization of thymus cellularity in CD3eD/D; eM-tg mice also indicated that the mutant CD3e protein could restore surface expression of pre-TCR complexes on immature DN thymocytes and that these complexes could transduce signals sufficient to promote the transition of DN thymocytes to the DP stage. To explore this issue further, we examined the phenotype of DN thymocytes obtained from CD3eD/D; eM-tg mice. As shown previously (15) and in Fig. 1 C, DN thymocytes are arrested at the CD442 CD251 stage in CD3eD/D mice. The observation that mu- tations in several distinct genes that are required either for 914 CD3e-mediated Signals in T Cell Development