The CD3‐zeta and CD3‐eta polypeptides are two of the components of the T cell antigen receptor (TCR) which contribute to its efficient cell surface expression and account for part of its transducing capability. CD3‐zeta and CD3‐eta result from the alternative splicing of a single gene designated CD3‐zeta/eta. To evaluate the role of these subunits during T cell development, we have produced mice with a disrupted CD3‐zeta/eta gene. The analysis of thymocyte populations from the CD3‐zeta/eta‐/− homozygous mutant mice revealed that they have a profound reduction in the surface levels of TCR complexes and that the products of the CD3‐zeta/eta gene appear to be needed for the efficient generation and/or survival of CD4+CD8+ thymocytes. Despite the almost total absence of mature single positive thymocytes, the lymph nodes from zeta/eta‐/− mice were found to contain unusual CD4+CD8‐ and CD4‐CD8+ single positive cells which were CD3‐. In contrast to the situation observed in the thymus, the thymus‐independent gut intraepithelial lymphocytes present in zeta/eta‐/− mice do express TCR complexes on their surface and these are associated with Fc epsilon RI gamma homodimers. These results establish an essential role for the CD3‐zeta/eta gene products during intrathymic T cell differentiation and further emphasize the difference between conventional T cells and thymus‐independent gut intraepithelial lymphocytes.

Fas/APO-1 is a member of the tumor necrosis factor receptor family of proteins that induces apoptosis when cross-linked with monoclonal antibody (mAb) or with its physiological ligand. Recently, both a perforin-based and a Fas-based mechanism have been proposed to account for T cell-mediated cytotoxicity. In the present study we used a murine CD8+ cytotoxic T lymphocyte (CTL) clone (KB5 C20) specific for H-2Kb and a T cell receptor (TcR)-negative variant of the same clone (2005-D4) to test (i) whether the same cell can exert both cytotoxic effector mechanisms and (ii) the role of TcR engagement in the induction of Fas-based cytotoxicity. We demonstrate that both the TcR+ and TcR- clones were able to express the Fas ligand after stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and that TcR engagement of the KB5.C20 clone by means of antigen-bearing cells or of its anticlonotypic mAb (Désiré-1), which leads to Ca(2+)-dependent, presumably perforin-based, cytotoxicity, was also able to induce Fas-based cytotoxicity. In addition, using inhibitors we investigated the signal transduction pathway(s) involved in the induction of Fas-based cytotoxicity and expression of the Fas ligand mRNA in the CTL clones. The involvement of src-like protein tyrosine kinases (PTK) in Fas ligand induction through TcR engagement, was strongly suggested by inhibition with the src-like PTK inhibitor herbimycin A. Inhibition of Fas ligand induction by genistein, a more general TPK inhibitor, even upon stimulation by PMA plus ionomycin, suggested the possible involvement of PTK activities downstream of protein kinase C (PKC) in Fas ligand induction in CTL. Finally, the implication of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in Fas ligand induction was demonstrated by the partial inhibition of Fas ligand induction with cyclosporin A. Thus, in CTL clones, Fas ligand expression is inducible by TcR engagement through a pathway similar to that involved in expression of some lymphokine genes.