Analysis of adhesion molecoles-mediated signal transduction in T cell activation by superantigen.

最近，我们报道了PMA或CD28交联物与超抗原和细胞因子传递的信号协同作用，诱导抗原提呈细胞(APC)缺失的T细胞增殖。提示蛋白激酶C(PKC)的激活参与了CD28交联介导的共刺激信号转导通路。我们研究了CD28交联剂对PKC早期激活的影响。首先，在自体APC、PMA或CD28交联剂存在下，PKC的特异性抑制剂calphostin C抑制PKC活性，抑制纯T细胞对来自A组链球菌的超抗原pep M5的反应。在体外，PKC检测在一定的细胞激活条件下，PKC从细胞质转移到质膜，在质膜上被激活。为了确定PKC是否通过CD28刺激信号转导通路被转位，用PMA或CD28交联剂刺激APC耗竭的静息T细胞，然后用本身对PKC活性没有影响的PEPM5+APC条件培养液(SUP)进行共刺激。在刺激后45min内检测胞浆和胞膜部分的PKC活性。与PMA不同，CD28单独交联物不能诱导膜相关PKC活性的增加。然而，在PEP M5+SUP存在的情况下，CD28交联导致膜相关PKC活性增加，30min后活性增加3倍，45min时活性下降。PEP M5+SUP单独作用对膜PKC活性无明显影响，即使在PKC活性高峰30min时，CD28交联物也能增强超抗原诱导的信号并激活PKC。

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 CInhibition 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 assayUnder 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.