INDUCTION OF ACTIVATOR PROTEIN (AP)-1 AND NUCLEAR FACTOR-KAPPA-B BY CD28 STIMULATION INVOLVES BOTH PHOSPHATIDYLINOSITOL 3-KINASE AND ACIDICSPHINGOMYELINASE SIGNALS

A major obstacle in understanding the signaling events that follow CD2 8 receptor ligation arises from the fact that CD28 acts as a costimulu s to TCR engagement, making it difficult to assess the relative contri bution of CD28 signals as distinct from those of the TCR. To overcome this problem, we have exploited the observation that activated human T cell blasts can be stimulated via the CD28 surface molecule in the ab sence of antigenic challenge; thus, we have been able to observe the r esponse of normal T cells to CD28 activation in isolation. Using this system, we observed that CD28 stimulation by B7-transfected CHO cells induced a proliferative response in T cells that was not accompanied b y measurable IL-2 production. However, subsequent analysis of transcri ption factor generation revealed that B7 stimulation induced both acti vator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappa B) complex es, but not NF-AT. In contrast, engagement of the TCR by class II MHC/ superantigen, either with or without CD28 ligation, resulted in the in duction of NF-AT, AP-1, and NF-kappa B as well as IL-2 production. Usi ng selective inhibitors, we investigated the signaling pathways involv ed in the CD28-mediated induction of AP-1 and NF-kappa B, This reveale d that NF-kappa B generation was sensitive to chloroquine, an inhibito r of acidic sphingomyelinase, but not to the phosphatidylinositol 3-ki nase inhibitor, wortmannin. In contrast, AP-1 generation was inhibited by wortmannin and was also variably sensitive to chloroquine. These d ata suggest that in activated normal T cells, CD28-derived signals can stimulate proliferation at least in part via NF-kappa B and AP-1 gene ration, and that this response uses both acidic sphingomyelinase and p hosphatidylinositol 3-kinase-linked pathways.