Activation of T cell hybridomas induces a G1/S cell cycle block and apoptosis. We isolated a variant of the 2B4.11 T cell hybridoma that, when activated via the TCR, produced IL-2 and underwent growth inhibition but did not die. Analysis of a variety of cell surface molecules revealed that the variant cell line, termed VD1, expressed very low levels of Fas compared to the wild type cells. Unlike 2B4.11 cells, VD1 cells were not killed by Fas ligand (FasL)-bearing effector cells. To determine if Fas is involved in activation-induced apoptosis, two different reagents that specifically bind Fas without killing the T cell hybridomas, a monoclonal antibody and a soluble Fas:Fc chimeric molecule, were added to activated T cell hybridomas. Both treatments prevented activation-induced apoptosis in a dose-dependent manner, but had no effect on IL-2 production or growth inhibition. Northern blot analysis revealed that unactivated 2B4.11 cells expressed negligible levels of FasL mRNA, but transcripts were detectable as early as 2 h after activation and continued to increase up to 4-6 h after activation. Anti-TCR induced activation of 2B4.11 cells in the presence of a TCR- 2B4.11 variant resulted in death of the unactivated "bystander" cells, which was inhibited by anti-Fas antibodies. Finally, treatment of T hybridoma cells with 9-cis retinoic acid or glucocorticoids, which are known to prevent activation-induced T cell apoptosis, inhibited the up-regulation of FasL. We conclude that up-regulated expression of FasL and its subsequent interaction with Fas accounts for the apoptotic response of T cell hybridomas to activation, and that retinoic acid and corticosteroids inhibit activation-induced apoptosis by preventing up-regulation of FasL.

Ribosome biogenesis has been associated with regulation of cell growth and cell division, but the molecular mechanisms that integrate the effect of ribosome biogenesis on these processes in mammalian cells remain unknown. To study the effect of impaired ribosome functions in vivo, we conditionally deleted one or two alleles of the 40S ribosomal protein S6 gene in T cells in the mouse. While complete deletion of S6 abrogated T-cell development, hemizygous expression did not have any effect on T-cell maturation in the thymus, but inhibited the accumulation of T cells in the spleen and lymph nodes, as a result of their decreased survival in the peripheral lymphoid organs. Additionally, TCR-mediated stimulation of S6-heterozygous T cells induced a normal increase in their size, but cell cycle progression was impaired. Genetic inactivation of p53 tumor suppressor rescued development of S6-homozygous null thymocytes and proliferative defect of S6-heterozygous T cells. These results demonstrate the existence of a p53-dependent checkpoint mechanism that senses changes in the fidelity of the translational machinery to prevent aberrant cell division or eliminate defective T cells in vivo. Failure to activate this checkpoint response could potentially lead to a development of pathological processes such as tumors and autoimmune diseases.[Keywords: S6 ribosomal protein; ribosome biogenesis; cell growth; cell proliferation; checkpoint] Supplemental material is available at http://www.genesdev.org.

The zeta (zeta) chain plays a central role in T cell antigen receptor assembly and signal transduction. From previous work in murine T cell hybridomas we have inferred that the zeta subunit is limiting in receptor assembly. Partial receptors made in excess of zeta are assembled in the endoplasmic reticulum, transported through the Golgi, but then rapidly and efficiently degraded in lysosomes. zeta would therefore seem to play a unique role in targeting receptors from the Golgi to the cell surface. To determine directly whether zeta limits receptor assembly we have reconstituted a zeta‐deficient T cell line by transfection of the murine zeta cDNA. Transfection results in restoration of expression of surface T cell receptor. In addition, increasing zeta expression results in a commensurate increase in the survival of previously excess subunits. This is reflected in an increased surface expression of complete receptors. Finally, transfection of the zeta cDNA fails to produce detectable zeta‐eta heterodimers. The implications of these findings with regard to receptor assembly, and the relationship between zeta and eta, are discussed.

T cell hybridomas that express zeta zeta, but not zeta eta, dimers in their T cell receptors (TCRs) produce interleukin-2 (IL-2) and undergo an inhibition of spontaneous growth when activated by antigen, antibodies to the receptor, or antibodies to Thy-1. Hybridomas without zeta and eta were reconstituted with mutated zeta chains. Cytoplasmic truncations of up to 40% of the zeta molecule reconstituted normal surface assembly of TCRs, but antigen-induced IL-2 secretion and growth inhibition were lost. In contrast, cross-linking antibodies to the TCR activated these cells. A point mutation conferred the same signaling phenotype as did the truncations and caused defective antigen-induced tyrosine kinase activation. Thus zeta allows the binding of antigen/major histocompatibility complex (MHC) to alpha beta to effect TCR signaling.