The fas/fas-ligand system is an important receptor-ligand system that regulates cell death in the immune system. Expression of functional fas ligand on activated T lymphocytes induces death of lymphocytes expressing fas and thus limits proliferation of antigen specific T and B cells. In contrast mutation of fas ligand to a nonfunctional form leads to a syndrome of lymphoproliferation and immunodeficiency due to the accumulation of lymphocytes. Recently, it was observed that expression of fas ligand on tissue transplants conferred protection from rejection, presumably by inducing death of alloreactive lymphoid populations. This project explores the feasibility of inducing tolerance to tissue allografts and xenografts by engineered expression of fas ligand on tissues for transplantation. In the first phase of the project, human and murine fas ligand are being targeted to professional antigen presenting cells (APC) such as Langerhans cells by cloning into an expression vector containing the MHC class II Ealpha promoter and constructing transgenic mice with this construct. Thus, professional APCs in all tissues should express fas ligand and transplantation of organs such as skin onto allogeneic or xenongeneic animals will be performed to test the ability of such organs to survive and induce tolerance. We will take advantage of mice in whom the vast majority of T cells express a unique receptor for alloantigen to assess the effect of expression of fas ligand on alloantigen expressing tissue. Controls will consist of a mutated fas ligand construct which is functionally inert or the vector alone. The second part of the project explores the feasibility of inducing tolerance to tissue allografts or xenografts by directly inoculating the tissue with the gene for fas ligand via gene gun. We have cloned the gene for human or for murine fas ligand into a mammalian expression vector, PCI, which has been used in gene gun protocols to induce immunity to viral antigens. In this protocol, a segment of skin from a donor animal will be """"""""shot"""""""" with fas ligand DNA and transplanted onto allogeneic animals to assess for effects on graft rejection.
