The long term goal of this project is to elucidate characteristics of, regulation of, and functional links between physiological processes in natural killer (NK) cells and cells bound by NK cells (target cells) which underlie the biological consequences of the NK-target cell interaction. The immediate goals of proposed studies are to investigate, in both NK and target cell, the multiple mechanisms of temporal changes in intracellular pH (pHi) and concentration of ionized Ca2+ ([Ca2+]i), two cellular functions likely to be associated with, and determinative of, physiological processes leading to target cell cytolysis. To accomplish these goals, we propose to use NK cells isolated from human peripheral blood and human cell lines, differing in lineage and sensitivity to NK cell-mediated lysis, as target cells to: i) make correlated temporal measurements of changes in NK and target cell [Ca2+]i and pHi which are initiated as a consequence of NK- target cell adhesion. Complementary measurements will be made by flow cytometry (to discern heterogeneity and kinetics of parameter changes in large populations of NK-target cell conjugates) and imaging cytometry (to obtain more detailed and spatially resolved kinetics information in isolated conjugates); ii) sort conjugates in the flow cytometer on the basis of NK-target cell [Ca2+]i and pHi response patterns. The association between response pattern and target cytolysis will be evaluated in single cell cytotoxicity assays of conjugates sorted onto agarose-coated slides; and iii) determine effects upon conjugation-associated temporal changes in [Ca2+]i and pHi of exposing NK and target cells to conditions which modulate cytolytic function (cytokines, bioactive mononucleotides, alkylating agents), defined cellular transmembrane ion flows (HCO3, Na+, Ca2+, Cl-, H+), and pHi (acidification, alkalinization). Integration of results form these experiments will identify patterns of change in [Ca2+]i and pHi which result from NK-target cell adhesion; how these patterns vary (frequency, amplitude, kinetics) as a function of manipulating cytolytic activity and target cell properties (lineage, NK-sensitivity); what patterns are associated with occurrence and non-occurrence of target cytolysis; and mechanisms by which NK and target cells govern [Ca2+]i and pHi fluctuation patterns under these various experimental conditions. The proposed studies will thus provide a more comprehensive view of physiological processes by which NK cells and target cells influence the outcome of this potentially cytolytic interaction which will further the understanding of the NK cell role in biological processes central to human health (i.e., lysis of tumors and virus-infected cells, regulation of B cells and developmental processes in thymus and marrow), and thereby provide a rational basis for health-related therapeutic NK cell manipulations.
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