Molecular Immunology. Vol. 34. No. 3, zyxwvutsrqponmlkjihg pp. 221-226, 1991 Pergamon PII: SO161-5890(97)00027-8 Q 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0161-5890/97 $17.00 + 0.00 EVIDENCE FOR PHOSPHATIDYLINOSITOL 3-KINASE- DEPENDENT T CELL ANTIGEN RECEPTOR (TCR) SIGNAL TRANSDUCTION MARK EXLEY* and LYUBA VARTICOVSKIQ *Department of Hematology/Oncology, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Av., Boston, MA 02215, U.S.A.; TDepartments of Medicine and Biomedical Research, St. Elizabeth’s Hospital, 736 Cambridge Street, and Tufts University, Boston, MA 02135, U.S.A. (First received 30 December 1996; accepted in revised,form 14 February 1997) Abstract-Recent evidence implicates PI 3-kinase in TCR signal transduction. The fungal metabolite wortmannin is a specific inhibitor of PI 3-kinase both in vitro and in vivo when used at nanomolar concentrations. Therefore, we examined the effect of wortmannin on stimulation of primary T cells and T cell lines. Wortmannin had a dose-dependent inhibitory effect on TCR-dependent primary T cell proliferation with zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA IC,, in the nanomolar range. Furthermore, activation of T cell lines independently of antigen presenting cells and, therefore of any CD28 co-stimulatory signaling, was also sensitive to wortmannin. As expected, phorbol ester stimulation bypassed PI 3-kinase signal transduction. Importantly, the effect of wortmannin correlated with inhibition of activation of PI 3-kinase in stimulated T cells. The earliest step in T cell activation, tyrosine kinase activation, was not significantly affected by wortmannin. We conclude that a wortmannin-sensitive enzyme, probably PI 3-kinase, acting downstream of tyrosine kinases, but independently of the phorbol ester activated pathway. is necessary for stimulation of T cells via the TCR, and that this requirement is independent of any role of PI 3-kinase in co-stimulation via CD28 coreceptor. PI 3-kinase is most probably involved in generation of 3-phosphorylated lipid products, and is not merely an adaptor. 0 1997 Elsevier Science Ltd. Key words: phosphatidylinositol3-kinase, TCR, T cell, wortmannin. INTRODUCTION Several signal transduction pathways are activated by interaction of the T cell antigen receptor (TCR) CD3 complex with antigen peptide/Major Histocompatibility Complex on antigen-presenting cells (Weiss and Littman, 1994; Terhorst zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA et al., 1996). The initiating event is tyro- sine phosphorylation by a src-family kinase (lck or fyn) and ZAP 70 (Samelson and Klausner, 1992; Perlmutter et al., 1993; Rudd et al., 1994; Weiss and Littman, 1994). T cell activation results in a substantial increase of basal Tyr phosphorylation of multiple substrates including CD3-y, 6, E, and { and kinase association (Samelson and Klausner, 1992; Weiss and Littman, 1994; Terhorst et al., 1996). Further biochemical events culminate in T cell division and effector functions such as IL-2 secretion and cytolysis. Analogous activation events occur in other $Author to whom correspondence should be addressed. Abbreviations: IL-Z, interleukin-2; ITAM, immuno-receptor tyrosine-based activation motif; PI, phosphatidylinositol; PIP,, PI 3,4,5_phosphate; P-Tyr, phosphotyrosine; PBL, per- ipheral blood leukocytes; PHA, phytohemagglutinin-P; PMA, phorbol myristic acid; SED, staphylococcal entero- toxin D; TCR, T cell antigen receptor; WM, wortmannin. hemopoietic cells (Perlmutter et al., 1993; Reth, 1994; Cambier, 1995; Jouvin et al., 1995). Signal transduction is based upon recruitment of specific SHf-containing sig- naling molecules by phosphorylated antigen receptor components. The signal transduction function has been localized to immuno-receptor tyrosine-based activation motifs (ITAM) which contain two Tyr residues phos- phorylated upon activation (Samelson and Klausner, 1992; Rudd et al., 1994; Weiss and Littman, 1994; Fla- swinkel et al., 1995; Cambier, 1995; Terhorst et al., 1996; Wange and Samelson, 1996). On TCR activation, the membrane distal [ ITAM associates with the SHC: Grb- 2mSOS complex (Ravichandran et al., 1993). Conversely, we found a specific activation-dependent association of phosphatidylinositol3-kinase (PI 3-kinase) with the phosphorylated membrane proximal 5 ITAM (Exley et al., 1994) and more recently with the E ITAM (deAos et al., manuscript in preparation). A role for PI 3-kinase in TCR-dependent T cell activation was also suggested by Tyr phosphorylation, and association with the activated TCR and downstream kinases (Thompson et al., 1992; Exley et al., 1994; Carrera et al., 1994; Rudd et al., 1994) and TCR-dependent accumulation of PI 3- kinase products (Ward et al., 1992; Exley et al., 1994; Ueda et al., 1995). Similarly, activation of the B cell 221