2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent immunosuppressive agents known, yet its cellular targets in the immune system and molecular mechanisms remain to be clarified. A gene regulatory protein, the Ah receptor (AhR), mediates the actions of TCDD. The goals of our research are to define the cellular and molecular targets of TCDD that lead to thymic atrophy, and determine how these events relate to its overall action on the immune system. Once these targets and actions are rigorously defined in young adult animals, future aims will extend these studies to perinatal development since this period appears to be exquisitely sensitive to TCDD. We have hypothesized that TCDD-elicited thymic atrophy and suppression of cell-mediated immunity is due, in part, to an effect on early T-cell development. We identified bone marrow (BM) as a target tissue, and established molecular markers for the action of TCDD on this tissue, and have shown that TCDD alters functional prothymocyte activity. We further hypothesize that TCDD, via the AhR, alters prothymocyte activity by modulating the expression of critical genes necessary for the differentiation and/or maturation of pre-T stem cells. Our continuing investigations will identify the direct cellular targets for TCDD, determine the role of the AhR, and begin to define the genes modulated. Using additional stem cell markers and high speed flow cytometry, we will characterize the phenotype of the affected stem cell populations(s) in BM. The goal of these studies is to more specifically define the hematopoietic progenitor cell(s) affected, and thus the stage of lymphopoiesis that is most sensitive to TCDD. By utilizing a developed in vivo BM-thymus reconstitution model and mouse strains that have arrested T-cell development, we will determine whether the affected BM stem populations have an inability to seed the thymus or undergo thymus- directed maturational processes. Using a combination of genetic and in vitro culture systems, we will determine if TCDD directly affects pre-T precursor populations or stromal elements which assist in the self- renewal, growth and differentiation of stem cell populations. Additional studies will utilize a combination of immunohistochemistry, in situ hybridization, and molecular biological techniques to determine if the AhR is localized to particular stem and/or stromal BM cell populations, and if AhR activation to a transcriptionally active form occurs in these cells. Finally, we have shown that estrogen, like TCDD, affects lymphoid stem cell development. Additional studies will determine if these effects occur by similar cellular and molecular pathways.
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