REGULATION OF NUCLEAR FACTOR-KAPPA-B AND ACTIVATOR PROTEIN-1 ACTIVITIES AFTER STIMULATION OF T-CELLS VIA GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED LY-6A E/

Cross-linking of glycosylphosphatidylinositol-anchored proteins, inclu ding mouse L gamma-GA/E, leads to IL-2 secretion and T cell activation , whereas engagement of Ly-GA/E uniquely inhibits IL-2 production indu ced via TCR. However, little is known concerning the molecular mechani sm by which glycosyphosphatidylinositol-anchored proteins regulate IL- 2 expression. In this study, we have examined the ability of an anti-L y-GA/E mAb to regulate transcription factors controlling IL-2 expressi on. Stimulation of EL4J(L gamma-6E). A 4 cells with anti-CD3 epsilon o r anti-L gamma GA/E mAbs induced nuclear factor (NF)-kappa B p65-p50 ( RelA/pSO) and AP-l (Fos/Jun) binding activities and increased nuclear factor of activated T cells (NF-AT) activity, whereas octamer-binding factor and NF-gamma levels were stable. Cyclic AMP response element bi nding protein and T cell-specific factor-1 (ct) activities were select ively enhanced by anti-CD3 epsilon, but not by anti-L gamma GA/E, whic h suggests that signaling via the TCR and L gamma-6 were not identical . Costimulation of these cells with both mAbs produced substantially r educed levels of AP-1, NF-AT, and, especially, NF-kappa B p65-p50 wher eas cyclic AMP response element binding protein and T cell-specific fa ctor-1 (a) were induced to a level seen after stimulation by anti-CD3 epsilon. The inducibility of the Il-2 enhancer in vivo and the contrib ution of individual transcription factors for this induction were asse ssed with use of reporter chloramphenicol acetyltransferase constructs containing the Il-2 enhancer or oligomerized binding sites for transc ription factors. These experiments also demonstrated a key role for NF -kappa B and AP-1 in the transcriptional regulation of the IL-2 gene b y TCR- and Ly6A/E-mediated signaling. By using the 2B4.11 T cell hybri doma and a mutated variant, we revealed a crucial role for the xi-chai n in Ly6A/E-mediated activation of NF-kappa B.