application) Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that frequently involves the kidney causing approximately 1% of all cases of adult, and 5% of pediatric, end- stage renal failure in the USA every year. The MRL mouse strain, when combined with either a defect in Fas (MRL-1pr/1pr) or in Fas ligand (MRL- gld/gld), develops a disease resembling human SLE and also, in a broader sense, serves as a model of autoimmunity due to failure of peripheral tolerance. The long-term goal of this application is to understand the function and regulation of pathogenic antigen-specific autoreative CD4+ T lymphocytes (ART) in this model by: 1) isolating ART clones, ideally specific for nucleosomal determinants, and providing their pathogenicity in adoptive transfer experiments; these ARTs will be used as a source of the T cell receptor DNA from which a T cell receptor transgenic mouse will be constructed. The ART will be derived from double mutant 1pr/gld MRL mice (with combined defects of Fas and Fas ligand) in order to facilitate these adoptive transfer experiments; in additional to conventional antigen presenting cells (APC), rheumatoid factor expressing transgenic B cells pulsed with immune complexes will be used as a source of APC; 2) developing a mouse transgenic for the alpha and beta chains of the T cell receptor of the selected pathogenic ART clone using appropriate molecular techniques to obtain rearranged V-alphaJ-alpha and VDJ-beta sequences, inserting these into appropriate vectors and establishing founders by blastocyst injection of the constructs; 3) characterizing T cell education, function and recirculation in the TCR transgenic mouse developed; this will be done by analyzing thymic selection, TCR-transgene expression, in vitro reactivity, in vivo disease-inducing properties of the transgenic T cell and in vivo sites of interaction of the transgenic T cell with autoantibody-producing B cells. This project should provide insights into basic mechanisms of autoimmunity relevant not only to SLE but also to other immunologically mediated renal disease in which autoreactive T cells play a pathogenic role. It will also serve as a valuable training vehicle whereby the applicant will extend his expertise in cellular immunology and acquire new understanding and skills in state of the art molecular biology, transgenic technology and immunohistochemistry.
