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 C1- 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29CA047401-01
Application #
3458923
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1988-05-05
Project End
1993-04-30
Budget Start
1988-05-05
Budget End
1989-04-30
Support Year
1
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Virginia
Department
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Haverstick, D M; Gray, L S (1993) Increased intracellular Ca2+ induces Ca2+ influx in human T lymphocytes. Mol Biol Cell 4:173-84
Haverstick, D M; Gray, L S (1992) Lymphocyte adhesion mediated by lymphocyte function-associated antigen-1. I. Long term augmentation by transient increases in intracellular cAMP. J Immunol 149:389-96
Densmore, J J; Szabo, G; Gray, L S (1992) A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes. FEBS Lett 312:161-4
Haverstick, D M; Sakai, H; Gray, L S (1992) Lymphocyte adhesion can be regulated by cytoskeleton-associated, PMA-induced capping of surface receptors. Am J Physiol 262:C916-26
Haverstick, D M; Gray, L S (1992) Lymphocyte adhesion mediated by lymphocyte function-associated antigen-1. II. Interaction between phorbol ester- and cAMP-sensitive pathways. J Immunol 149:397-402
Hewlett, E L; Gray, L; Allietta, M et al. (1991) Adenylate cyclase toxin from Bordetella pertussis. Conformational change associated with toxin activity. J Biol Chem 266:17503-8
Anderson, G; Gray, L S; Mintz, P D (1991) Red cell survival studies in a patient with anti-Tca. Am J Clin Pathol 95:87-90
Haverstick, D M; Engelhard, V H; Gray, L S (1991) Three intracellular signals for cytotoxic T lymphocyte-mediated killing. Independent roles for protein kinase C, Ca2+ influx, and Ca2+ release from internal stores. J Immunol 146:3306-13
Gray, L S; Huber, K S; Gray, M C et al. (1989) Pertussis toxin effects on T lymphocytes are mediated through CD3 and not by pertussis toxin catalyzed modification of a G protein. J Immunol 142:1631-8