T lymphocytes play a vital role in host immune defense against viruses, bacteria, fungi and tumor cells. Activation of T lymphocytes involves the binding of the T cell antigen receptor to its specific antigen. Upon binding, a series of biochemical events take place at the level of the plasma membrane that ultimately produce the activation of genes that control cell proliferation. However, large gaps in our knowledge currently exist about the activation events that take place in the cytoplasm that link the membrane and gene activation events. The goal of this proposal is to better understand the role of rapidly phosphorylated cytosolic proteins involved in the cell activation of T lymphocytes. Little more than the size of many phosphorylated cytosolic proteins is currently known. As a consequence, large gaps in our understanding of cell activation currently exist. Recently developed molecular techniques in the form of antisense oligodeoxynucleotides provide a powerful tool to explore early cellular activation events. Expression of specific genes can be selectively inhibited by antisense oligodeoxynucleotides, thus avoiding many of the pitfalls of using nonspecific inhibitory compounds. Upon introduction of antisense oligomers into the cell, mRNA binds to the oligomers in a sequence-specific manner to terminate the translation of the desire protein. It is the specific aim of this proposal to investigate which phosphorylated cytosolic proteins control early activation events in anti-CD3-stimulated T lymphocytes by 1.) isolating the proteins that are phosphorylated early in cell activation by gel electrophoresis and sequencing those proteins by automated protein microsequencing, 2.) using antisense oligodeoxynucleotides, generated by an oligonucleotide synthesizer, to attempt to inhibit the activation of the cells, and 3.) cloning and sequencing the genes responsible for encoding these proteins. Understanding the process of immune cell activation has great potential medical relevance. The lack of tumor killing by tumor-infiltrating lymphocytes may be understood to arise from a suppression of T lymphocyte activation. In addition, we may better understand why states of immune unresponsiveness exist in certain congenital immunodeficiencies in spite of the presence of large numbers of apparently mature lymphocyte populations. Finally, this proposal may extend the knowledge of how antisense oligodeoxynucleotides may be used to inhibit cell activation, potentially important to the treatment of cancer.
