We have recently demonstrated that human peripheral blood mononuclear (PBM) cells, when cultured in vitro with Listeria monocytogenes, generate high levels of non-MHC restricted cytotoxic activity and produce TNF-alpha and IFNs. The overall goal of this proposal is to further investigate the human cell-mediated immune functions which are activated by Listeria and to elucidate what virulence factor(s) and mechanism(s) are involved in such immune activation.
The specific aims of this proposal are: 1) To characterize the cell populations present in human PBM cells which can be activated by Listeria monocytogenes to generate non-MHC-restricted cytotoxic activity. 2) To determine if Listeria monocytogenes can induce lymphokine production by human PBM cells and by different T cell lines and/or clones and to determine what role these lymphokines play in Listeria-induced non-MHC restricted cytotoxic activity. 3) To characterize the bacterial cell surface associated components and/or secreted products which are involved in Listeria monocytogenes activation of human PBM cells. 4) To determine if Listeria cell surface components and/or secreted products which induce cytokine production or non-MHC restricted cytotoxic activity can be recognized as antigens by human Listeria-specific T cells. We will characterize the cells mediating non-MHC restricted cytotoxic activity by selective cell depletion using antibody conjugated magnetic beads, antibody and complement lysis, and/or by flow cytometry. Cytokines produced by Listeria activated PBM cells will be identified using molecular biological techniques, by specific RIAs/ELISA assays, or by bioassays. Using cDNA for TNF, IFN, IL2, IL6 or specific oligonucleotide primers, we will quantitate specific MRNA present in different cell populations stimulated by Listeria. Cells producing cytokines will be characterized by in situ hybridization combined with immunocytochemistry. We will investigate different proteins derived from Listeria as well as peptidoglycan, lipoteichic and teichoic acids, listeriolysin O, and other secreted and cell associated components isolated using standard biochemical techniques for their ability to activate different PBM cell populations. The studies proposed in this application represent a first step at investigating how different human cell-mediated immune effector functions respond to Listeria.
