SummaryWe have developed a model of peripheral in vivo T cell tolerance that is induced by administration of the protein superantigen staphylococcal enterotoxin B (SEB). Rather than activating V08+ T cells, in vivo administration of SEB induced in them a profound state of anergy. This was shown by their failure to proliferate to subsequent in vitro restimulation with SEB or to antiV08 antibodies. This unresponsiveness was V08 specific since T cells from SEB-immunized mice responded normally to other antigens. 8 d after SEB administration, there was no reduction in the number of Va8+ T cells or in the intensity of V08 T cell receptor (TCR) expression . Although a portion of the V08+ T cells from SEB-primed mice were able to express interleukin 2 receptors (IIr2Rs), they failed to proliferate in response to exogenous IL2, indicating they were defective in their IL-2 responsiveness. 2-4 wk after SEB administration, there was a reduction of -50% in the number of V08+ cells in immunized compared with control animals . There was, however, no reduction in the level of TCR expression on the remaining V08+ cells. These data demonstrate that proteins that activate T cells in vitro in a V/3-specific manner can induce a state of anergy in peripheral T cells in vivo and may possibly further mediate clonal deletion in a portion of the tolerized cells.

Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g., SDF-1) stimulation inactivates ERM proteins, causing their release from the plasma membrane and dephosphorylation. SDF-1–mediated inactivation of ERM proteins is blocked by phospholipase C (PLC) inhibitors. Conversely, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels by activation of PLC, expression of active PLC mutants, or acute targeting of phosphoinositide 5-phosphatase to the plasma membrane promotes release and dephosphorylation of moesin and ezrin. Although expression of phosphomimetic moesin (T558D) or ezrin (T567D) mutants enhances membrane association, activation of PLC still relocalizes them to the cytosol. Similarly, in vitro binding of ERM proteins to the cytoplasmic tail of CD44 is also dependent on PIP2. These results demonstrate a new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM protein biology, namely hydrolysis-mediated ERM inactivation.

The zeta subunit of the T cell antigen receptor (TCR) exists primarily as a disulfide-linked homodimer. This receptor subunit is important in TCR-mediated signal transduction and is a substrate for a TCR-activated protein tyrosine kinase. The zeta chain was found to undergo ubiquitination in response to receptor engagement. This posttranslational modification occurred in normal T cells and tumor lines. Both nonphosphorylated and phosphorylated zeta molecules were modified, and at least one other TCR subunit, CD3 delta, was also ubiquitinated after activation of the receptor. These findings suggest an expanded role for ubiquitination in transmembrane receptor function.

Elf-1, a member of the E 26-specific transcription factor family with a predicted molecular mass of 68 kDa, is involved in the transcriptional regulation of several hematopoietic cell genes. We demonstrate that Elf-1 exists primarily as a 98-kDa form in the nucleus and as an 80-kDa form in the cytoplasm. Phosphorylation and O-linked glycosylation contribute to the increased posttranslational molecular mass of Elf-1. The 98-kDa Elf-1 is released from the cytoplasm tethering retinoblastoma protein and moves to the nucleus, where it binds to the promoter of the TCR ζ-chain gene. Finally, the cytoplasmic 98-kDa form enters the proteasome pathway and undergoes degradation. In conclusion, different forms of Elf-1 are the products of posttranslational modifications that determine its subcellular localization, activity, and metabolic degradation.

B-cell receptor (BCR)-induced activation of phospholipase C-gamma1 (PLCgamma1) and PLCgamma2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCgamma activation, the mechanism coupling PLCgamma to the BCR remains undefined. The role of PLCgamma1 SH2 and SH3 domains at different steps of BCR-induced PLCgamma1 activation was examined by reconstitution in a PLCgamma-negative B-cell line. PLCgamma1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCgamma1 with the adapter protein, BLNK. Forcing PLCgamma1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCgamma1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCgamma1 activation.