INHIBITION OF THE T-CELL RECEPTOR-MEDIATED SIGNAL-TRANSDUCTION BY MICROINJECTION OF ANTI-LCK MONOCLONAL-ANTIBODY INTO T-CELLS

Engagement of T-cell receptor (TcR)/CD3 complexes on T-cells rapidly provokes tyrosine phosphorylation of cellular proteins, which is thought to be an essential step to the following events of T-cell activation. p56(lck), a member of src-related, non-receptor type protein tyrosine kinases, is expressed predominantly in lymphocytes. Accumulating data suggest that p56(lck) is one of the kinases responsible for TcR-mediated protein tyrosine phosphorylation. To investigate the role of p56(lck) in TcR-signaling in detail, we injected anti-lck monoclonal antibody (mAb), MOL171 or MOL294, both specifically suppress Lck kinase activity in vitro, into Jurkat T-cells by the erythrocyte-ghost procedure in order to block the activity of p56(lck). In Jurkat cells injected with anti-lck mAb, intracellular Ca2+ mobilization induced by TcR-stimulation was markedly reduced in comparison with control mouse IgG-injected samples. This block of Ca2+ influx seems to be specific for TcR-signaling because anti-lck mAb-injection did not cause significant suppression of phytohaemagglutinin-induced Ca2+ increase. Furthermore, injection of anti-Lck mAb inhibited TcR-mediated protein tyrosine phosphorylation of 100 kDa protein and phospholipase C gamma 1. These results confirm that p56(lck) is an indispensable element of TcR-signaling and p100 and phospholipase C gamma 1 are strongly presumed to be candidates for substrates for p56(lck).