Fas ligand (FasL) is a transmembrane protein originally described as a proapoptotic molecule inducibly expressed on cytotoxic T cells. Recent studies have shown that FasL expression can also trigger an inflammatory response, depending on the nature and status of the Fas+ target population. As such, it is evident that FasL expression must be stringently regulated. One aspect of this regulation is the ability of the membrane-bound form of FasL to be cleaved by a metalloproteinase; such cleavage rapidly reduces the level of FasL cell expression and releases a soluble protein that serves as an antagonist. Thus the overall impact of FasL expression is a balance between the membrane-bound and soluble forms.FasL can also be constitutively expressed by non-lymphoid tissues and, in some of these cases, FasL expression has been linked to the immune privilege of certain tissues or immune evasion of certain tumors. The significance of cleavage at these sites are unknown. The functional properties of transfected cell lines that express either wildtype FasL (wtFasL), membrane-only FasL (mFasL), or soluble-only FasL (sFasL) have been compared, and cells expressing mFasL were found to be remarkably more potent effectors than their wtFasL counterparts. For example, mFasL cells kill Fas+ target cells 5-10 times more effectively than wtFasL cells; mFasL cells induce greater neutrophil extravasation into the peritoneum than wtFasL cells; and mFasL lymphoma cells injected into syngeneic mice via the """"""""immunoprivileged"""""""" anterior chamber of the eye are rejected and induce long-term immunity. Based on these observations, the goals of the current application will be two-fold. First, to analyze the cell types responsible for FasL-triggered tumor rejection and long term immunity and to further explore potential applications of mFasL expression to tumor immunotherapy regimens and secondly, to determine how cleavage moderates the function of FasL expressed by lymphoid and non-lymphoid tissues.This analysis will be facilitated by the use of a knock-in mouse strain rendered incapable of FasL cleavage as a result of gene-targeted deletion of the FasL metalloproteinase site. These studies will help to assess the feasibility of clinical applications of forced FasL expression and they should further reveal the significance of FasL expression by non-lymphoid tissues.
