T lymphocyte activation is initiated by antigen (Ag) binding to the T-cell receptor in the context of the appropriate Ia molecule. How this initial event signals activation-associated gene expression is not known. Calcium and hydrogen ion fluxes and changes in cyclic nucleotide levels have been observed in many eukaryotic cells stimulated with soluble mitogens, suggesting that these biochemical changes may be important in signal transduction. In order to investigate intracellular biochemical events associated with Ag-specific T-cell activation, and MHC- restricted, bovine insulin-specific T-T hypbridoma has been developed in this laboratory. In this biologically relevant model, intracellular calcium ion concentration ((Ca+2)i) rises and intracellular pH (pHi) increases when antigen binds to the T-cell receptor. The proposed research will examine how these intracellular biochemical changes modulate the T-cell response to antigen. The mechanism and physiologic significance of intracellular alkalinization will be investigated in Ag-activated T-cells by inhibiting Na/H antiport function with pharmacologic agents or by genetic mutation. Interleukin-2 (IL-2) production will be used as a measure of T-cell effector function. Down-regulation of protein kinase C (PK-C) with low concentrations of phorbol esters will determine if activation of PK-C is necessary for cellular activation. By comparing two-dimensional electrophoresis patterns of 32P-labeled cytoplasmic phosphoproteins form ionophore-stimulated cells vs. Ag-activated cells, we will determine if increased pHi can substitute for PK-C-mediated phosphorylation as a signal for T-cell activation. Changes in cyclic nucleotide levels will be measured in Ag-stimulated T- cells; modulation of cyclic nucleotide levels by pHi changes will be investigated, as well as reciprocal modulation of Ag-specific activation by cyclic nucleotides. The kinetics of Il-2 mRNA accumulation and the appearance of nuclear transcripts for IL-2 will be examined in cells treated with Ca+2 or H+ ionophores or cyclic nucleotide analogs to determine if changes in (Ca+2)i, pHi, and/or (cAMP)i can effect transcriptional control of IL-2 genes. By suggesting a molecular basis for biochemical regulation of T lymphocyte activation, these studies should contribute significantly to an understanding of immunopathologic mechanisms in neoplastic and autoimmune diseases, and may provide insight into new methods of controlling the undesired cellular activation in these diseases.
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| Adams, B S; Leung, K Y; Hanley, E W et al. (1991) Cloning of R kappa B, a novel DNA-binding protein that recognizes the interleukin-2 receptor alpha chain kappa B site. New Biol 3:1063-73 |
