The long term goal of this proposal is to arrive at a comprehensive understanding of the mechanism of signal transduction and lethal hit delivery in cytolytic T lymphocytes (CTL). CTL are an ultimate effector cell in the cellular arm of the immune response and are involved in host defense from neoplasia and viral illness as well as graft rejection and graft-vs-host disease. Thus, the mechanism by which these cells are activated to produce these effects is of fundamental biomedical importance.
The Specific Aims of this proposal are: 1. to relate the functionally important transmembrane fluxes of Ca2+, K+ and Cl- to one another and to changes in membrane potential and intracellular pH to determine mechanisms of control of these events and the consequences of their occurrence, and; 2. to examine the role of cAMP and inositol polyphosphates (IP) in lytic function and to relate changes in the intracellular concentrations of these second messengers to ion fluxes as well as to determine the possible role of cAMP in control of protein phosphorylation and antigen induced morphologic changes in CTL. These studies will use fluorescence spectroscopy, flow cytofluorimetry, and high resolution cinemicrography to document the involvement of ionic species in CTL mediated lysis and to relate these events to morphological changes which accompany the lytic process. Additionally, various pharmacological agents will be used to inhibit cytolysis by distinct mechanisms. Phosphoprotein analysis, measurement of intracellular cAMP and IP as well as analysis of G protein involvement will be conducted to relate various inhibitory manipulations to known regulatory pathways. These observations will be synthesized into a pathway leading from target cell binding to cytolysis.
