Antigen/Fas ligand genetic therapy for the induction of specific peripheral tolerance by T cell apoptosis

Fas ligand (FasL, FasL) induces apoptosis in activated T cells which have upregulated Fas (CD95) expression through the process termed activation induced cell death (AICD). We postulated that co-expression of an antigen and FasL within individual antigen presenting cells would lead to antigen specific activation of T cells and to their consequent deletion by FasL mediated AICD. A DNA-gelatin coacervate containing transferrin cell ligand, calcium, and the lysosomatropic agent chloroquine, a formulation previously shown to achieve high level transfection of immune and muscle cells in vivo, was used to co-deliver plasmids encoding FasL and antigen. Mice developed a strong cytolytic T cell response to β-gal when injected with DNA encoding β-galactosidase (LacZ) model antigen, either as naked DNA or DNA nanoparticles, but fail to respond when there was concomitant injection of nanoparticles containing both the LacZ and murine FasL DNA vectors. This loss of T cell response was systemic, specific for β-gal, complete when nanoparticles were administered before antigen challenge, and decreased the T cell response from prior immunization with LacZ DNA. In effect, this “tolerization” injection induced antigen specific peripheral tolerance to the model antigen in study mice. After this demonstration of tolerance induction, two model systems were used to test the system for efficacy in prevention of tissue rejection and treatment of autoimmune diseases.; The CT26 cell line transfected with and stably expressing the β-gal antigen is rapidly rejected by an immune response to β-gal when injected into a mouse. This cell line was used to test the possibility that tolerization to rejection antigens would protect a transplanted tissue. Mice injected with the cell line alone rapidly developed a T cell response to β-gal and rejected the transplanted cell, where those first treated with βgal/FasL tolerization lacked a β-gal specific immune response and tissue remained protected; demonstrating that the system can induce protection of a slightly different self tissue from immunologic destruction.; The NOD mouse, which develops a disease similar to human Type 1 diabetes, was used as a model to test for efficacy of tolerization in a multi-factor, clinically relevant system. Mice were treated with GAD65/FasL nanoparticles before onset of disease. Tests for T cell function demonstrated that reactivity to GAD65 was completely abrogated by the tolerization. Nonetheless, mice developed diabetes at a rate almost identical to untreated mice. These results indicate that complex diseases involving multiple reactive antigens most likely require tolerance induction to more than one antigen.; Overall the antigen/Fas ligand gene therapy system induces the deletion of specific T cells, and may have utility in treatment of graft rejection and autoimmune diseases.