Antigens, mitogens and lymphokines initiate a series of events which lead to lymphocyte activation. T cell activation is determined by measuring the terminal events of RNA, DNA and Interleukin-2(IL-2) synthesis. An early nuclear event which precedes RNA and DNA synthesis and is associated with lymphocyte activation is the induction of polyamine synthesis. The initial enzyme in this pathway is ornithine decarboxylase (ODC). The transfer of mitogenic signals from the membrane to the nucleus of a cell is a prerequisite for the induction of ODC. This proposal is directed at identifying the enzymes which transfer the various signals associated with T cell activation, i.e. antigen, mitogen, Interleukin-1 (IL-1) Interleukin-2 (IL-2), from the membrane to the nucleus (Aim 1). These enzymes, which activate polyamine synthesis, will be investigated with metabolic inhibitors and stimulators (Aim 2) to study their interaction and possible control.
Aim 3 will examine the role of lymphokine cell surface receptors in directing which enzyme pathways become activated following stimulation with lymphokines, and Aim 4 will investigate the influence of the T cell immunosuppressant Cyclosporin A on enzyme activation and polyamine synthesis.
These Aims will be accomplished by using biochemical techniques established in normal and transformed cells which measure enzyme activation (cAMP-dependent protein kinase I and II, cAMP-independent, calcium-calmodulin dependent and calcium-phospholipid-dependent protein kinases) with immunological techniques to assess T lymphocyte activation (ODC induction, DNA, synthesis and Il-2-production). Human antigen specific T cell clones and T cell tumor lines will be stimulated to activate the various protein kinase enzymes. Protein kinase activation will be correlated with polyamine synthesis measured by ODC induction. T lymphocyte activation will be measured by DNA (3H-thymidine) and RNA (3H-uridine) synthesis and IL-2-production (proliferation of the IL-2-dependent cell line CTLL-2) as terminal events of T cell activation. Specific metabolic inhibitors and stimulators will be used to evaluate the interaction of protein kinase activation in T lymphocytes with the subsequent effect on polyamine synthesis. CsA, which will specifically block antigen-driven but not IL-2-driven T cell activation, will also be used to study these early enzymatic events. These investigations may yield new information on the normal enzyme mechanisms which initiate polyamine synthesis and T cell activation as examined at the intracellular level.
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