The overall objective of our studies is to investigate the underlying mechanisms by which NK cells initiate their cytotoxic activity against sensitive target cells. It has been implicated that cytolysis by NK cell is a secretory process and more recently soluble cytotoxic mediators have been reported by us and others and have been postulated to be involved in the lethal hit stage of lysis. We have formulated a model which accounts for most known characteristics of NK CMC and in which natural killer cytotoxic factors (NKCF) play a role. We propose to examine the activation or trigger stage of NK by target cells (TC) resulting in target cell cytotoxicity. Our preliminary findings have identified a mechanism involving the polyphosphoinositol hydrolysis cascade and protein kinase activation. Accordingly, this proposal will examine pathways of NK activation and initiate studies on the nature of structures of NK involved in the trigger. Our studies will be done in parallel in both the NK CMC reaction and NKCF production. Studies will be done with purified sub-populations of NK and at the single cell level.
Our specific aims are (1) To characterize the initial pathway of activation of NK cells by NK target cells. This will include the role of GTP binding proteins, phosphodiesterase activation, the phosphatidylinositol pathway, Ca++ mobilization, protein kinase C activation and phosphorylation, and membrane depolarization. These studies will be correlated with secretion of NKCF or antigenically TNF-like molecules in NKCF; (2) To define the nature of NK effector cell membranes involved in activation and determine whether recognition/binding alone is sufficient for activation. This will be accomplished by using non-lytic conjugating NK cells, target cell variants and blocking monoclonal antibodies. The studies above will be done with a method developed by us to purify NK killer and non-killer cells separated by sorting on a flow cytometer. Furthermore, activation will be correlated with secretion using sensitive bioassay and radioimmunoassay techniques for NKCF and TNF.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA043121-02
Application #
3185063
Study Section
Experimental Immunology Study Section (EI)
Project Start
1987-07-01
Project End
1991-12-31
Budget Start
1989-01-01
Budget End
1989-12-31
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Jewett, A; Gan, X H; Lebow, L T et al. (1996) Differential secretion of TNF-alpha and IFN-gamma by human peripheral blood-derived NK subsets and association with functional maturation. J Clin Immunol 16:46-54
Jewett, A; Bonavida, B (1996) Target-induced inactivation and cell death by apoptosis in a subset of human NK cells. J Immunol 156:907-15
Jewett, A; Bonavida, B (1994) Pentoxifylline suppresses interleukin-2-mediated activation of immature human natural killer cells by inhibiting endogenous tumor necrosis factor-alpha secretion. J Clin Immunol 14:31-8
Bonavida, B; Braquet, M; Lebow, L T et al. (1994) Qualitative and quantitative analysis of subpopulations of cytotoxic effector cells by flow cytometry. J Lipid Mediat Cell Signal 9:19-25
Lebow, L T; Jewett, A; Bonavida, B (1993) Killer cell recruitment and renewal capacity of purified cytolytic and noncytolytic human peripheral blood natural killer cell subsets. J Immunol 150:320-9
Bonavida, B; Lebow, L T; Jewett, A (1993) Natural killer cell subsets: maturation, differentiation and regulation. Nat Immun 12:194-208
Jewett, A; Bonavida, B (1993) Pivotal role of endogenous TNF-alpha in the IL-2-driven activation and proliferation of the functionally immature NK free subset. Cell Immunol 151:257-69
Safrit, J T; Bonavida, B (1992) Hierarchy of in vitro sensitivity and resistance of tumor cells to cytotoxic effector cells, cytokines, drugs and toxins. Cancer Immunol Immunother 34:321-8
Bonavida, B (1991) Immunomodulatory effect of tumor necrosis factor. Biotherapy 3:127-33
Lebow, L T; Bonavida, B (1990) Purification and characterization of cytolytic and noncytolytic human natural killer cell subsets. Proc Natl Acad Sci U S A 87:6063-7

Showing the most recent 10 out of 13 publications