| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1994: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
Recently, we reported that either PMA or CD28 cross-linking synergizes with signals delivered by superantigen and cytokines to induce to proliferation of antigen presenting cells (APC)-depleted T cells. It is suggested that protein kinase C (PKC) activation is involved in the costimulatory signal transduction pathway mediated by CD28 cross-linking. We have examined the effects of CD28 cross-linking on early activation of PKC.
First ; a specific inhibitor of PKC,calphostin C
Inhibition of PKC activity by calphostin C suppressed the response of pure T cells to pep M5, superantigen derived from group A Streptococci strain, in the presence of either autologous APC,PMA,or CD28 cross-linking. It was observed only when calphostin C was added within an first hour after stimulation.
Second ; in vitro PKC assay
Under a certain condition of cell activation, PKC is translocated from cytoplasm to plasma membrane where it becomes activated. To determine whether PKC is translocated by stimulation of the signal transduction pathway via CD28, APC-depleted resting T cells were stimulated with either PMA or CD28 cross-linking followed by costimulation with pep M5 plus APC-conditioned medium (SUP) which per se had no effect on PKC activity. PKC activity in both cytosolic and membrane fractions was measured within 45 min after stimulation. Unlike PMA,CD28 cross-linking alone failed to induce an increase in membrane-associated PKC activity. However, in the presence of pep M5 plus SUP,CD28 cross-linking resulted in an increase in membrane-associated PKC activity that reached a three-fold increase in activity by 30 min after addition of stimuli and declined by 45 min. Pep M5 plus SUP alone had no significant effect on membrane PKC activity even when measured at 30 min, the peak of PKC activation.
We conclude that CD28 cross-linking augments superantigen-induced signals and activates PKC.
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