Figure 4. Positive selection of thymocytes from CD3eD/D; e-tg and CD3eD/D; eM-tg mice ex- pressing H-Y– or P14-transgenic TCRs. (A) Comparison of thy- mocytes and lymph node T cells from H-Y TCR–transgenic/ CD3eD/D; e-tg and H-Y TCR- transgenic/CD3eD/D; eM-tg fe- males. Two-color plots show CD4/CD8 staining profiles on total or T3.701 (gated) thy- mocytes and lymph node cells. Numbers in quadrants indicate the percentage of cells within that quadrant. Histograms show staining of total thymocytes or CD81 lymph node T cells with the H-Y clonotype–specific anti- body, T3.70. (The percentage of T3.70hi cells and the gate used for the T3.701 two-color plots is also shown.) Results are repre- sentative of four separate experi- ments. (B) Comparison of thy- mocytes and lymph node T cells from P14 TCR–transgenic/ CD3eD/D; e-tg and P14 TCR-transgenic/CD3eD/D; eM-tg mice. Two-color plots show CD4/CD8 staining profiles on total or Va21 (gated) thymocytes and lymph node cells. Numbers in quadrants indicate the percentage of cells within that quadrant. Histograms show staining of total thymocytes or CD81 lymph node T cells with the P14 TCR a chain–reactive mAb Va2. (The percentage of Va2hi cells and the gate used for the Va21 two-color plots is also shown.) Results are representative of two separate experiments. contained similar numbers of phenotypically (Fig. 4 and data not shown) and functionally (Fig. 3 C) mature T3.70hi CD8-SP thymocytes. To determine if the effect of the eM-tg was specific to cells that express the H-Y TCR, we examined develop- ment and selection of thymocytes expressing a different MHC class I–restricted TCR, P14. Thymocytes that express the P14 TCR are also positively selected in the H-2Db background, leading to the generation of CD8-SP/Va2hi thymocytes and peripheral T cells (17). Examination of thymocytes and peripheral (lymph node) T cells from P14 TCR-tg/CD3eD/D; eM-tg mice revealed a phenotype nearly identical to that of P14 TCR-tg/CD3eD/D; e-tg mice (i.e., mice of both genotypes contained similar num- bers of CD8-SP/Va2hi thymocytes and peripheral T cells) (Fig. 4 B). Thus, the generation and survival of CD8-SP T cells that express the P14 TCR did not appear to be com- promised in CD3eD/D; eM-tg mice. Although other explanations remain possible, these re- sults are most consistent with the notion that CD3e signals are required for the survival of naive H-Y TCR1 but not P14 TCR1 T cells. Recent studies have shown that self- MHC is required for long-term survival of naive T cells (25, 26). However, to our knowledge, the data presented here are the first that directly implicate TCR signaling in T cell survival. Our results could reflect a unique require- ment for CD3e-mediated signals in T survival; however, they are also consistent with the idea that CD3e contrib- utes quantitatively to the TCR signaling response. A gen- eralized defect in T cell survival was not observed in CD3eD/D; eM-tg mice but instead was seen only in the H-Y TCR–transgenic background. Significantly, the self- ligand that positively selects and presumably maintains the survival of peripheral H-Y TCR1 T cells is thought to bind with especially low affinity to the TCR (27, 28). Thus, the survival of H-Y TCR1 CD8-SP T cells but not P14 TCR1 CD8-SP T cells may critically depend upon the presence of the entire complement of TCR-ITAMs. Consistent with this idea, the generation of H-Y TCR1 CD8-SP T cells but not P14 TCR1 CD8-SP T cells by positive selection was found to be absolutely dependent upon TCR z chain ITAMs (9, 10). Moreover, P14 TCR1 CD8-SP T cells but not H-Y TCR1 CD8-SP T cells are triggered to undergo homeostatic proliferation when transferred to H-2Db T cell–depleted hosts, again suggest- ing that H-Y TCR1 CD8-SP T cells bind with lower af- finity to self-ligands (28, 29). Another possibility that can- not presently be ruled out is that the mutant CD3e chain expressed in CD3eD/D; eM-tg mice is not inert but instead inhibits TCR signaling. An inhibitory role for z chains that contain partially (mono)phosphorylated ITAMs has been postulated based on data from in vitro studies (30). However, an inhibitory effect was not observed when a z chain containing mutated ITAMs capable of becoming mono- but not diphosphorylated was expressed in trans- genic mice (9). Additional experiments will be required to determine the precise function of TCR signaling in gen- eral and CD3e signals in particular in regulating T cell sur- vival. We thank Dr. Ronald Germain for critical reading of the manu- script and Victoria Saunders for technical assistance. 918 CD3e-mediated Signals in T Cell Development