Cytotoxic T cells (CTL) are one of the cornerstones of the immune system. There is therefore much interest in understanding the mechanism by which they lyse targets, which is the overall goal of this proposal. Although significant strides have been made in our understanding of CTL lysis many questions remain open. Adsorption of CTL to targets via receptors triggers polarization of cytoskeletal elements in the CTL. This leads to reorientation of the Golgi apparatus (GA) towards the target binding site and secretion of channel forming proteins -perforins (PF) - which are thought to cause target cell lysis. Recent evidence suggest s that this sequence of events established with in vitro CTL clones does not operate in vivo induced CTL. Evidence is also accumulating that target lysis by channel formation may not be the universal mechanism of CTL lysis and that the target itself plays an active role in its lysis. We propose to explore several aspects of the concurrent models for CTL lysis in in vivo induced CTL, because they have not yet acquired abnormal properties in vitro. CTL will be highly purified and tested whether they show GA reorientation when bound to targets because this reaction is thought to signal proper target recognition. Next CTL will be tested as to their ability to insert PF channels into erythrocyte membranes. To induce this reaction we will use bifunctional anti-target anti-T cell receptor antibody conjugates. If CTL fail to insert channels into erythrocytes we will test whether removal of nuclei from targets inhibits target lysis. In drug inhibition studies we will examine whether continued lytic activity of the CTL on one hand and lytic susceptibility of the target on the other require protein synthesis prior to CTL-target binding. Preliminary data support the view that targets participate in CTL induced lysis. To explore how targets participate in lysis we propose several approaches, one of which is based on the ability of protein synthesis inhibitors to interfere with CTL lysis. Another is based on the concomitant occurrence of resistance to CTL lysis and resistance to glucocorticoids in one cell line. We propose to examine whether there indeed exists a correlation between glucocorticoid resistance and CTL sensitivity. This would provide support for a common pathway for both mechanisms of cell lysis. We also propose to select CTL resistant targets to explore whether they acquire resistance to PF and/or glucocorticoids. These variants will provide the tool to study the postulated suicide pathway. Finally, we will explore whether in vivo induced CTL change their mode of lysis upon prolonged culture in vitro and activate pathways of cell mediated lysis that have been described for cloned CTL.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA039623-06
Application #
3178839
Study Section
Immunobiology Study Section (IMB)
Project Start
1985-07-01
Project End
1993-11-30
Budget Start
1990-12-01
Budget End
1991-11-30
Support Year
6
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Southern California
Department
Type
Schools of Medicine
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Nemoto, E; Stohlman, S; Dennert, G (1996) Release of a glycosylphosphatidylinositol-anchored ADP-ribosyltransferase from cytotoxic T cells upon activation. J Immunol 156:85-92
Jamora, C; Dennert, G; Lee, A S (1996) Inhibition of tumor progression by suppression of stress protein GRP78/BiP induction in fibrosarcoma B/C10ME. Proc Natl Acad Sci U S A 93:7690-4
Wang, J; Nemoto, E; Dennert, G (1996) Regulation of CTL by ecto-nictinamide adenine dinucleotide (NAD) involves ADP-ribosylation of a p56lck-associated protein. J Immunol 156:2819-27
Nemoto, E; Yu, Y; Dennert, G (1996) Cell surface ADP-ribosyltransferase regulates lymphocyte function-associated molecule-1 (LFA-1) function in T cells. J Immunol 157:3341-9
Wang, J; Nemoto, E; Kots, A Y et al. (1994) Regulation of cytotoxic T cells by ecto-nicotinamide adenine dinucleotide (NAD) correlates with cell surface GPI-anchored/arginine ADP-ribosyltransferase. J Immunol 153:4048-58
Wang, J; Stohlman, S A; Dennert, G (1994) TCR cross-linking induces CTL death via internal action of TNF. J Immunol 152:3824-32
Sugawara, S; Takeda, K; Lee, A et al. (1993) Suppression of stress protein GRP78 induction in tumor B/C10ME eliminates resistance to cell mediated cytotoxicity. Cancer Res 53:6001-5
Takeda, K; Dennert, G (1993) The development of autoimmunity in C57BL/6 lpr mice correlates with the disappearance of natural killer type 1-positive cells: evidence for their suppressive action on bone marrow stem cell proliferation, B cell immunoglobulin secretion, and autoimmune sy J Exp Med 177:155-64
Sugawara, S; Kaslow, H R; Dennert, G (1993) CTX-B inhibits CTL cytotoxicity and cytoskeletal movements. Immunopharmacology 26:93-104
Kikly, K; Dennert, G (1992) Evidence for a role for T cell receptors (TCR) in the effector phase of acute bone marrow graft rejection. TCR V beta 5 transgenic mice lack effector cells able to cause graft rejection. J Immunol 149:3489-94

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