MOLECULAR MECHANISMS OF T-LYMPHOCYTE ACTIVATION

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The objective of this study is to investigate the coupling mechansism between the T cell receptor (TCR)/CD3 complex of lymphocytes and phospholipase C (PLC), the key enzyme in the inositol phospholipid (InsPL) hydrolysis pathway. This pathway generates second messengers that may be critical in inducing T cell activation. An understanding of the mechanism of lymphocyte activation may offer a basis for designing immunomodulatory strategies targeted to critical elements of the signal transducion pathway. A strategy of cell permeabilization has been developed in our laboratory which allows access to micromolecules (e.g., nucleotide, peptides, etc.) to the intracellular enviromnment of lymphocytes, while maintaining coupling of the TCR/CD3 complex with PLC. By using this strategy, we have obtained information on the regulation of InsPL hydrolysis and on the stoichiometry of the assoication of a src kinase, fyn, with polypetides of the CD3 complex. By the use of this strategy, we will seek information on the mechanism of control of PLC activity. Coprecipitation using anti-PLC Ab and src-family products (fyn, lck.ves, etc.) will be used. Transient or "weak" interactions will be "stabilized" by using homobifunctional chemical cross-linker in permeabilized cells followed by immunoprecipitation. This strategy was previously used successfully to obtain stoichionetry data of CD3/fyn interation. By using inhibitors of protein tyrosine phosphate phosphatase (including peptides which may function as potential competitive inhibitors, such as phosphorylated fyn or lck peptides) in intact and permeabilized cells, attention will be paid to the role of these enzymes, including the CD45 phosphatase, in modulating insPL hydrolysis. Since previous evidence suggests heterogeneity in signal transduction among different T cell subsets, attention will be paid to potential differences and their functional implications. AN initial attempt will be made to construct T cell clones defective in PLC (PLC- y1) by genetically "knocking out" the enzyme.