We have previously developed a method for the transfer of genes into nontransformed human T lymphocyte clones. Stable high level gene expression was achieved by transfections with a self-replicating Epstein-Barr virus episomal replicon. By including anti-sense CD8 in this vector, we have created a clonal phenocopy with specific loss of expression of CD8. The overall aim of the work proposed in this application is to produce variants of T cell clones with altered gene expression via our transfection protocol and to investigate the functional effects of these specific alterations. The T cell molecules targeted for both anti-sense and plus-sense alteration are CD2, CD4, CD8, CD28, CD45 (T200), and interleukin 2. The genes represent a broad spectrum of important T lymphocytic characteristics including activation, cytotoxicity, lymphokine secretion, immunoregulation, and subsets. Specific alteration of molecules in T cell clones by our transfection procedure will permit the analysis of the functions of these molecules. The specific functions that will be assessed include proliferation, cytotoxicity, help for immunoglobulin production, lymphokine secretion, acquisition of surface activation markers, intracellular ionized calcium concentrations after activation, phosphoinositide metabolism after activation, and regulatory cell-to-cell interactions among T cells. Both antigen-specific and nonspecific stimuli will be used to activate the cells. The feasibility of our approach and the potential for obtaining new insights has been demonstrated in initial work on CD8. Alternative methods of investigating the functions of cellular proteins cannot replace our methods, but they serve as complementary techniques that will corroborate and challenge our own interpretations.
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