TCR Signaling Responses Are Intact or Mildly Impaired in CD3eD/D; eM-tg Mice. To assess whether the TCR com- plexes expressed on thymocytes from CD3eD/D; eM-tg mice can transduce signals similar to those normally elicited by TCR cross-linking, we first examined if TCR engage- ment led to the activation of downstream signaling path- ways. Several early events in the TCR activation cascade, such as the phosphorylation of z chain (Fig. 2 A), ZAP-70, and LAT (linker for activation of T cells; data not shown), were unaffected in CD3eD/D; eM-tg mice. These results were not unexpected, as ZAP-70 is known to be activated following its association with phosphorylated z chain ITAMs after TCR engagement, and z chain expression is unaltered in CD3eD/D; eM-tg mice. Activation-induced calcium responses were also indistinguishable in thy- mocytes from CD3eD/D; e-tg and CD3eD/D; eM-tg mice (data not shown). Mature T cell responses in CD3eD/D; eM-tg mice were examined by analyzing the ability of peripheral T cells to proliferate and produce IL-2 after cross-linking of the TCR and CD28. Although no significant differences were observed under conditions of maximal TCR stimulation, T cells from CD3eD/D; eM-tg mice exhibited impaired prolif- erative responses (Fig. 3 A) and cytokine responses (data not shown) when limiting concentrations of stimulating antibody were used (Fig. 3 A). Impaired proliferative re- sponses were also observed when proliferation was assessed by CFSE staining (Fig. 3 B). Together, these results dem- onstrate that although CD3e-mediated signals are not spe- cifically required for T cell maturation or mature T cell functional responses, they contribute quantitatively to TCR signaling. Thymocyte Selection and T Cell Survival in TCR-tg/ CD3eD/D; eM-tg Mice. To examine the role of CD3e signals in thymocyte selection, we bred the H-Y TCR transgene into the CD3eD/D; e-tg and CD3eD/D; eM-tg back- grounds. Positive selection of thymocytes in H-Y TCR- tg/H-2Db female mice results in the generation of CD8-SP thymocytes and T cells that express high levels of the H-Y clonotypic TCR (detected by mAb T3.70) (16). Positive selection of H-Y TCR1 thymocytes appeared uncompro- mised in H-Y TCR-tg/CD3eD/D; eM-tg mice when com- pared with H-Y TCR-tg/CD3eD/D; e-tg mice, as assessed by total thymocyte numbers and the number and percent- age of CD8-SP/T3.70hi cells in the thymus (Fig. 4 A). However, examination of peripheral lymphoid organs re- vealed a striking paucity of CD8-SP/T3.70hi T cells in H-Y TCR-tg/CD3eD/D; eM-tg mice (Fig. 4 A). Low num- bers of CD8-SP/T3.70hi T cells were consistently observed in both the lymph nodes and spleens of H-Y TCR-tg/ CD3eD/D; eM-tg mice regardless of the age of the mice tested (range 4 wk to 6 mo). Although CD8-SP/T3.70hi T cell numbers were markedly reduced in H-Y TCR-tg/ CD3eD/D; eM-tg mice, CD8-SP/T3.702 and CD4-SP T cell numbers were similar to or greater than those in H-Y TCR-tg/CD3eD/D; e-tg mice, indicating that the genera- tion and/or survival of CD8-SP/T3.70hi peripheral T cells was selectively compromised in H-Y TCR-tg/CD3eD/D; eM-tg mice. One possible explanation for the results obtained with H-Y TCR-tg/CD3eD/D; eM-tg mice is that T3.70hi CD8- SP thymocytes fail to complete the maturation process in the thymus. However, arguing against this, H-Y TCR-tg/ CD3eD/D; e-tg and H-Y TCR-tg/CD3eD/D; eM-tg mice 917 Sommers et al. Brief Definitive Report Figure 3. (A) Functional responses of T cells from CD3eD/D; e-tg and CD3eD/D; eM-tg mice. CD41CD82 T cells were purified from lymph nodes and cultured on plates precoated with anti- CD3e and varying amounts of anti-CD28 mAbs as indicated. After 40 h, cells were pulsed with [3H]thymidine and harvested. j, CD3eD/D; e-tg. m, CD3eD/D; eM-tg. Results shown are representa- tive of those obtained in five of six experiments (in one experiment, the proliferative responses of T cells from CD3eD/D; e-tg and CD3eD/D; eM-tg mice were not significantly different). (B) Analysis of proliferative responses by CFSE labeling. Exper- imental conditions were identical to those de- scribed in A except that cells were labeled with CFSE before stimulation. Histograms show CFSE staining on live gated CD41 cells. Results shown are representative of three individual experiments. (C) Proliferative responses of CD8-SP thymocytes from H-Y TCR-tg/CD3eD/D; e-tg and H-Y TCR-tg/CD3eD/D; eM-tg mice. CD42CD81 thy- mocytes were purified and cultured on plates with APCs prepared from female mice 1 peptide anti- gen (Smcy; KCSRNRQYL). After 40 h, cells were pulsed with [3H]thymidine and harvested. j, H-Y TCR-tg/CD3eD/D; e-tg. m, H-Y TCR-tg/ CD3eD/D; eM-tg. r, CD3e1/1.