SIGNAL TRANSDUCTION VIA THE T CELL RECEPTOR /CD3 COMPLEX

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The objective of this study is to determine the coupling mechanism between the T cell receptor (TCR) of T helper (Th) lymphocytes and phospholipase C (PLC), the key enzyme in the inositol phospholipid (InsPL) hydrolysis pathway. An understanding of the mechanism of lymphocyte activation represents the base for designing immunomodulatory strategies targeted to critical elements of the signal transduction pathway, based upon the information acquired. Preliminary findings: Effect of quanine nucleotide analogs. Exposure of murine Th cells permeabilized with streptolysin O (SLO) or tetanolysin (TL) to the non- hydrolyzable guanosine triphosphate (GTP) analog, guanosine-5'-0-(3- - thiotriphosphate) (GTPgammaS), resulted in inositol phosphate (InsP) generation. Similarly, perturbation of the TCR/CD3/ with the MoAb 145.2C11 (directed against the CD3 epsilon chain) resulted in InsPL hydrolysis by permeabilized cells. A role for a G-protein in TCR/CD3/ PLC coupling was further indicated by the inhibition of TCR/CD3-mediated InsPL hydrolysis by GDP, a quanine nucleotide analog that competes for GTP. Microelements and nucleotide requirement. InsP generation induced by either TCR/CD3 perturbation or GTPgammaS treatment showed similar Ca2+ dependence. An indication for a role of kinases in PLC activation was supported by the observation that ATP was strictly required for TCR/CD3-mediated InsPL hydrolysis, while only potentiated GTPgammaS- induced InsP generation. Other nucleotides (CTP, GDP, GTP, ITP) did not affect the response. Attempts will be made in the future to gain information on the nature of both the putative G-protein and tyrosine kinase involved in modulating PLC activity. A strategy of coprecipitation using anti-PLC Ab (PLC- gamma1 and PLC-beta) and/or anti-src and src-related oncogene products (fyn, lck, yes, etc.) will be used to this end. By taking advantage of permeabilized cells, transient, "weak" interaction could be stabilized by using homobifunctional chemical cross-linker followed by immunoprecipitation. Preliminary efforts using this techniques have been satisfactory. By using inhibitors of protein tyrosine phosphate phosphatase (sodium orthovanadate or molybdate) in intact and permeabilized cells, attention will be paid to the role of these enzymes, including the CD45 phosphatase, in modulating InsPL hydrolysis.