MRL mice homozygous for the lpr (lymphoproliferation) gene (MRL-lpr/lpr) develop lymphadenopathy, splenomegally and autoimmune disease. The lpr gene has not been mapped and is defined only by the autoimmune phenotype and the molecular and cellular basis underlying the lymphoproliferative autoimmune syndrome is unknown. Adult MRL-lpr/lpr mice develop a 100 fold increase in T cells in lymphoid organs. The majority of these T cells do not express CD4 or CD8 but do express a normal T-cell receptor alpha-beta heterodimer. Extensive studies of these mice suggest that T cells undergo abnormal thymic development resulting in a defect of self tolerance. This results in excessive production of autoreactive T cells that migrate from the thymus in large numbers to populate the spleen and lymph nodes. Despite the importance ascribed to production of autoreactive T cells in autoimmune disease of mice and humans, this process has not been directly demonstrated to occur, and no information is available to suggest the mechanism for production of autoreactive T cells. The present proposal will study a MRL-lpr/lpr mouse that expresses a transgenic self-reactive T- cell receptor alpha and beta chain on 90% of thymic and peripheral T cells to directly determine if self-reactive T cells exit the thymus in large numbers and contribute to autoantibody production and autoimmune disease. MRL-lpr/lpr mice that have been backcrossed to a transgenic mouse that expresses the T-cell receptor for the H-2D(b) restricted male H-Y antigen will be used for these experiments. The T-cell receptor beta chain uses the V(beta)8.2 region, detected by the anti-V(beta)8 antibody F23.1. This allows easy identification of the transgenic T-cell receptor by flow cytometry or immunohistochemistry. Expression of CD4, CD8 and the T-cell receptor will be studied during thymic development in transgenic MRL- lpr/lpr and control mice to determine the nature of the tolerance defect. Self-reactive T-cell subpopulations from thymus, lymph node, and spleen will be studied by cytotoxic and proliferation assays, and for the ability to escape clonal deletion or induction of anergy. BrdU labeling and bone marrow transfer experiment will precisely identify the developmental stage of abnormal tolerance induction. These experiments will help clarify the cellular mechanism leading to the loss of T-cell tolerance, and role of autoreactive T-cells in the autoimmune disease in MRL-lpr/lpr mice.
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